Compositions and methods for treating diabetes

ABSTRACT

The present invention provides an isolated population of cells containing an expressible nucleic acid encoding proinsulin containing a proinsulin cleavage site and a glucose-regulated expressible nucleic acid encoding a protease capable of cleaving the proinsulin cleavage site to produce insulin. The invention also provides an isolated population of cells which further express a hexosamine synthetic pathway enzyme. The invention additionally provides vectors containing an expressible nucleic acid encoding proinsulin containing a proinsulin cleavage site and a glucose-regulated expressible nucleic acid encoding a protease capable of cleaving the proinsulin cleavage site to produce insulin. The invention further provides a method of treating or preventing diabetes by implanting into an individual cells coexpressing proinsulin containing a proinsulin cleavage site and a glucose-regulated protease capable of cleaving the proinsulin cleavage site to produce insulin.

COMPOSITIONS AND METHODS FOR TREATING DIABETES

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/087,660, filed Jun. 2, 1998, and is incorporatedherein by reference.

[0002] This invention was made with government support under grantnumber DK43727 and DK50686 awarded by the National Institutes of Health.The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates generally to methods for thetreatment and prevention of diabetes and to the use of cells which canexpress insulin in a glucose-regulated manner for this treatment.

[0004] In an individual with normal regulation of blood glucose, thepancreatic hormone insulin is secreted in response to increased bloodsugar levels. Increased blood glucose generally occurs following a mealand results from insulin action on peripheral tissues such as skeletalmuscle and fat. Insulin stimulates cells of these peripheral tissues toactively take up glucose from the blood and convert it to forms forstorage. This process is also referred to as glucose disposal. Thelevels of blood glucose vary from low to normal to high throughout theday within an individual, depending upon whether the person is in thefasting, intermediate, or fed state. These levels are also referred toas hypoglycemia, euglycemia and hyperglycemia, respectively. In thediabetic individual, these changes in glucose homeostasis aredisregulated due to either faulty insulin secretion or action, resultingin a chronic state of hyperglycemia.

[0005] Diabetes mellitus is a common disorder, with a prevalence ofabout 4-5%. The risk of developing diabetes increases with increasedweight, with as many as 90% of adult onset diabetic patients beingobese. Therefore, due to the high incidence of obese adults, theincidence of adult onset diabetes is increasing worldwide. Diabetesmellitus is classified into three major forms. Type 2 diabetes is oneform and is also referred to as non-insulin dependent diabetes (NIDDM)or adult-onset diabetes. Type 1 diabetes is the second form and isreferred to as insulin-dependent diabetes (IDDM). The third type ofdiabetes is genetic and is due to mutations in genes controllingpancreatic islet beta (β) cell function. Although the diagnosis ofdiabetes is based on glucose measurements, accurate classification ofall patients is not always possible. Type 2 diabetes is more commonamong adults and type 1 diabetes dominates among children and teenagers.

[0006] Diabetes mellitus of both types 1 and 2 are associated with ashortened life expectancy as well as other complications such asvascular disease and atherosclerosis. Long-term management of diabetesto prevent late complications often includes insulin therapy regardlessof whether the patients are classified as type 1 or type 2. Type 1diabetes is an auto-immune disease which is associated with nearcomplete loss of the insulin producing pancreatic β cells. This loss ofβ cells results in insulin-dependence for life. Type 1 diabetes canoccur at any age and it has been estimated that about 1% of all newbornswill develop this disease during their lifetime.

[0007] Insulin is first synthesized as a precursor and cleaved throughthe action of proteases to form a mature, bioactive molecule thatconsists of two subunits covalently bonded together. Insulin functionand regulation have been studied in regard to both therapeutic andresearch applications. For example, insulin has been produced fortherapeutic purposes by recombinant expression using modified cells. Asa research tool, insulin was the first protein to be chemicallysynthesized and also has been used to study secretory pathways and theregulation of secretory mechanisms.

[0008] A widely used method of treatment for type 1 diabetes and to someextent type 2 diabetes has classically consisted of insulin maintenancetherapy. Such therapy in its simplest form requires the injection ofpurified or recombinant insulin into a patient following ingestion of ameal or at regular intervals throughout the day to maintain normal bloodglucose levels. These injections are required ideally at a frequency offour times per day. Although the above method of treatment provides somebenefit to the patient, this method of insulin therapy neverthelesssuffers from inadequate blood glucose control as well as requiring agreat deal of patient compliance.

[0009] Another method of treatment for type 1 diabetes includes the useof devices such as an insulin pump which allows for the scheduleddelivery of insulin. This method can be preferable to the methoddescribed above due to the need for less frequent injections. However,the use of an insulin pump therapy also has drawbacks in thatreplacement of a needle once every three days is still required. Similarto insulin maintenance therapy, the insulin pump method also does notachieve optimal glucose regulation as the delivery of insulin is notregulated in response to changes in blood glucose level. These methodsof treating diabetes are therefore burdensome as well as inadequate.Furthermore, although these methods can provide some benefit forreducing symptoms of diabetes, none have been completely effective overthe course of an average adult lifetime and none have been shown to beeffective in preventing this disease.

[0010] Various approaches of cell therapy for replacing bioactiveinsulin into a diabetic individual have been attempted. These includegene therapy approaches, immunotherapies and use of artificial β-cells.

[0011] In vivo gene therapy for insulin expression has included livertargeted retroviral-mediated transduction in rats and adenoassociatedvirus administration to mice (Kolodka et al., Proc. Natl. Acad. Sci. USA92:3293-3297 (1995); Sugiyama et al., Horm. Metab. Res. 29:599-603(1997)). However, these approaches did not provide glucose-regulatedinsulin delivery and have limited applications in patients.

[0012] Treatment for diabetes has also included studies attempting tomanipulate the immune system. In particular, to reverse theautoreactivity against the beta cell specific autoantigens, insulin orglutamic acid decarboxylase (GAD65) have been injected into youngdiabetes-prone NOD mice or BB rats (Kaufman et al., J. Clin. Invest.89:283-292 (1992); Kaufman et al., Nature 366:69-72 (1993); Bieg et al.,Diabetologia 40:786-792 (1997)). Reversal of autoreactivity against betacell specific autoantigens has also been attempted with nasaladministration of GAD65 peptides (Tian et al., J. Exp. Med.183:1561-1567 (1996)).

[0013] Genetic modification of pancreatic islet β-cells and generationof artificial beta cells are approaches for the treatment of diabetes bycell therapy (Becker et al., Methods Cell Biol. 43:161-189 (1994);Efrat, Diabetes Reviews 4:224-234 (1996); Newgard, Diabetes 43:341-350(1994); Gros et al., Hum. Gene Ther. 8:2249-2259 (1997); Taniguchi etal., J. Surg. Res. 70:41-45 (1997). Xenograft and even allogeneic celldelivery to express insulin require cell encapsulation to prevent hostimmune responses, and problems with cell survival and sustained insulindelivery have been identified (Kawakami et al., Cell Transplant.6:541-545 (1997); Wang et al., Nature Biotechnol. 15:358-362 (1997);Zhou et al., Am. J. Physiol. 274:C1356-C1362 (1998); Scharp et al.,Diabetes 43:1167-1170 (1994)).

[0014] Pancreatic and islet transplantation has also been attempted as atreatment for diabetes. Use of this treatment has shown limited successdue to the requirement for matched tissue from 2-5 adult donors perrecipient. This method has also lacked success due, in part, to thefailure of the transplanted tissue to maintain normal glucose-regulatedinsulin secretion and to remain viable over a reasonable period of time.

[0015] Thus, there exists a need for simple and more efficient methodsthat can regulate glucose homeostasis in a diabetic individual in a waythat more closely mimics a normal endogenous insulin response. Thepresent invention satisfies this need and provides additionaladvantages.

SUMMARY OF THE INVENTION

[0016] The present invention provides an isolated population of cellscontaining an expressible nucleic acid encoding proinsulin containing aproinsulin cleavage site and a glucose-regulated expressible nucleicacid encoding a protease capable of cleaving the proinsulin cleavagesite to produce insulin. The invention also provides an isolatedpopulation of cells which further express a hexosamine synthetic pathwayenzyme. The invention additionally provides vectors containing anexpressible nucleic acid encoding proinsulin containing a proinsulincleavage site and a glucose-regulated expressible nucleic acid encodinga protease capable of cleaving the proinsulin cleavage site to produceinsulin. The invention further provides a method of treating orpreventing diabetes by implanting into an individual cells coexpressingproinsulin containing a proinsulin cleavage site and a glucose-regulatedprotease capable of cleaving the proinsulin cleavage site to produceinsulin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows vectors expressing proinsulin, proinsulin andglucose-regulated furin, and glutamine:fructose-6-phosphateamidotransferase.

[0018]FIG. 2 shows the hematocrit analysis of rats implanted withprosthetic stomach grafts consisting of smooth muscle cells transducedwith either rat erythropoietin (Epo) or human adenosine deaminase (ADA).

[0019]FIG. 3 shows β-galactosidase (β-gal) expression and hematoxylinand eosin staining of cross sections of prosthetic vascular graftsconsisting of smooth muscle cells transduced with β-gal expressingvirus. The prosthetic grafts were implanted in the femoral artery ofdogs.

[0020]FIG. 4 shows the nucleotide and amino acid sequences of humanproinsulin cDNA (SEQ ID NOS:1 and 2, respectively; GenBank accession No.X70508; Chekhranova et al., Mol. Biol. 26:596-600 (1992)). The A-chainof human insulin corresponds to nucleotides 312-374 (SEQ ID NO:3) andamino acids 90-110 (SEQ ID NO:4). The B-chain of human insulincorresponds to nucleotides 117-206 (SEQ ID NO:5) and amino acids 25-54(SEQ ID NO:6).

DETAILED DESCRIPTION OF THE INVENTION

[0021] This invention is directed to cell populations and methods fortreating or preventing diabetes. The cell populations contain nucleicacids encoding proinsulin containing a proinsulin cleavage site and aprotease capable of cleaving the proinsulin cleavage site. The methodsof the invention are directed to implanting the above cell populationsinto diabetic individuals for the therapeutic production of insulin. Anadvantage of the cell populations and their use in treating diabetes isthat the proinsulin and protease are coexpressed, with expression of theprotease under glucose regulation. This regulated coexpression resultsin the amplification of secreted bioactive insulin in aglucose-responsive manner.

[0022] In one embodiment, implantable, non-endocrine cells such asvascular smooth muscle cells are constructed to express proinsulin and aprotease. These cells exhibit the ability to secrete high levels ofglucose-regulated mature insulin and are constructed by transduction ofthe cells with a three-gene retroviral vector. The three-gene retroviralvector contains elements required for the constitutive expression ofproinsulin and the glucose-regulated expression of the protease, furin.Regulated expression of furin is under the control of aglucose-responsive transforming growth factor α (TGFα) promoter andregulatory element. The retroviral vector also encodes for theselectable marker neomycin phosphotransferase, which allows for thepositive selection of cells transduced with and expressing the vector.Exposure of the population of transduced vascular smooth muscle cells toa high glucose environment results in secretion of bioactive insulin atlevels that are sufficient for treating diabetes.

[0023] In a second embodiment of the invention, the above describedsmooth muscle cells expressing glucose-regulated insulin are furthermodified to express the hexosamine biosynthetic pathway enzymeglutamine:fructose-6-phosphate amidotransferase. Expression of thisadditional gene further enhances expression of the protease, whichallows for enhanced processing of proinsulin to mature insulin.Implantation of these modified cells into the stomach wall of a diabeticanimal is achieved using a prosthetic graft made of a biocompatiblematerial such as polytetrafluoroethylene (PTFE). Glucose-regulatedinsulin expression from these smooth muscle cells engrafted into adiabetic individual allows for glucose homeostasis in that individualsufficient to provide therapeutic benefit.

[0024] As used herein, the term “diabetes” is intended to mean thediabetic condition known as diabetes mellitus. Diabetes mellitus is achronic disease characterized by relative or absolute deficiency ofinsulin which results in glucose-intolerance. The term is intended toinclude all types of diabetes mellitus, including, for example, type I,type II, and genetic diabetes. Type I diabetes is also referred to asinsulin dependent diabetes mellitus (IDDM) and also includes, forexample, juvenile-onset diabetes mellitus. Type I is primarily due tothe destruction of pancreatic β-cells. Type II diabetes mellitus is alsoknown as non-insulin dependent diabetes mellitus (NIDDM) and ischaracterized, in part, by impaired insulin release following a meal.Insulin resistance can also be a factor leading to the occurrence oftype II diabetes mellitus. Genetic diabetes is due to mutations whichinterfere with the function and regulation of β-cells.

[0025] Diabetes is characterized as a fasting level of blood glucosegreater than or equal to about 140 mg/dl or as a plasma glucose levelgreater than or equal to about 200 mg/dl as assessed at about 2 hoursfollowing the oral administration of a glucose load of about 75 g. Theterm “diabetes” is also intended to include those individuals withhyperglycemia, including chronic hyperglycemia and impaired glucosetolerance. Plasma glucose levels in hyperglycemic individuals include,for example, glucose concentrations greater than normal as determined byreliable diagnostic indicators. Such hyperglycemic individuals are atrisk or predisposed to developing overt clinical symptoms of diabetesmellitus.

[0026] As used herein, the term “treating” is intended to mean anamelioration of a clinical symptom indicative of diabetes. Ameliorationof a clinical symptom includes, for example, a decrease in blood glucoselevels or an increase in the rate of glucose clearance from the blood inthe treated individual compared to pretreatment levels or to anindividual with diabetes. The term “treating” also includes an inductionof a euglycemic response in the individual suffering from disregulatedhyperglycemia. Euglycemia refers to the range of blood glucose levelsclinically established as normal, or as above the range of hypoglycemiabut below the range of hyperglycemia. Therefore, a euglycemic responserefers to the stimulation of glucose uptake to reduce the plasma glucoseconcentration to normal levels. For most adults, this level correspondsto the range in concentration of about 60-105 mg/dL of blood glucose andpreferably between about ₇₀-100 mg/dL, but can vary between individualsdepending on, for example, the sex, age, weight, diet and overall healthof the individual. Effective treatment of a diabetic individual, forexample, would be a reduction in that individual's hyperglycemia, orelevated blood glucose levels, to normalized or euglycemic levels, withthis reduction directly resulting from secretion of insulin.Alternatively, effective treatment would be a reduction in fasting bloodglucose to levels less than or equal to about 140 mg/dL.

[0027] The term “treating” is also intended to include the reduction inseverity of a pathological condition or a chronic complication which isassociated with diabetes. Such pathological conditions or chroniccomplications are listed in Table 1 and include, for example, musclewasting, ketoacidosis, glycosuria, polyuria, polydipsia, diabeticmicroangiopathy or small vessel disease, atherosclerotic vasculardisease or large vessel disease, neuropathy and cataracts. TABLE 1Pathological Conditions Associated with Diabetes Kidney Glomerularmicroangiopathy Renal Diffuse glomeruloscierosis Nodularglomerulosolerosis (Kimmel- stiel-Wilson disease) Urinary infectionsAcute pyelonephritis Failure Necrotizing papillitis Emphysematouspyelonephritis Glycogen nephrosis (Armanni-Ebstein lesion) EyeRetinopathy Visual Nonproliferative retinopathy; capillaryMicroaneurysms, retinal edema exudates, and hemorrhages Proliferativeretinopathy: proliferation of small vessels, Failure hemorrhagefibrosis, retinal detachment Cataracts Transient refractive errors dueto osmotic changes in lens Glaucoma due to proliferation of vessels inthe iris Infections Nervous System Cerebrovascular atheroscleroticdisease: strokes, death Peripheral neuropathy; peripheral sensory andmotor cranial, autonomic Skin Infections: folliculitis leading tocarbuncles Necrobiosis lipoidica diabeticorum: due to microangiopathyXanthomas: secondary to hyperlipidemia Cardiovascular system Coronaryatherosclerosis: myocardial infarction, death Peripheralatherosclerosis: limb ischemia, gangrene Reproductive system Increasedfetal death rate (placental disease, neonatal respiratory distresssyndrome, infection) General Increased susceptibility to infectionDelayed wound healing

[0028] Additional complications also include, for example, a generalincreased susceptibility to infection and wound healing. The term“treating” is also intended to include an increase in the average lifeexpectancy of a diabetic individual compared to a non-treatedindividual. Other pathological conditions, chronic complications orphenotypic manifestations of the disease are known to those skilled inthe art and can similarly be used as a measure of treating diabetes solong as there is a reduction in the severity of the condition,complication or manifestation associated with the disease.

[0029] As used herein, the term “preventing” is intended to mean aforestalling of a clinical symptom indicative of diabetes. Suchforestalling includes, for example, the maintenance of normal levels ofblood glucose in an individual at risk of developing diabetes prior tothe development of overt symptoms of the disease or prior to diagnosisof the disease. Therefore, the term “preventing” includes theprophylactic treatment of individuals to guard them from the occurrenceof diabetes. Preventing diabetes in an individual is also intended toinclude inhibiting or arresting the development of the disease.Inhibiting or arresting the development of the disease includes, forexample, inhibiting or arresting the occurrence of abnormal glucosemetabolism such as the failure to transfer glucose from the plasma intothe cells. Therefore, effective prevention of diabetes would includemaintenance of glucose homeostasis due to glucose-regulated insulinexpression in an individual predisposed to a diabetic condition, forexample, an obese individual or an individual with a family history ofdiabetes. Inhibiting or arresting the development of the disease alsoincludes, for example, inhibiting or arresting the progression of one ormore pathological conditions or chronic complications associated withdiabetes. Examples of such pathological conditions associated withdiabetes are listed in Table 1.

[0030] As used herein, the term “implanting” is intended to mean theintroduction or transplantation of cells into an individual wherein thecells remain viable after implantation and maintain theirglucose-regulated insulin secretion for at least one stimulation ofglucose uptake. Implanting includes, for example, direct grafting of thecells, administration with other components such as matrix components,fragments or other molecules which facilitate adhesion of the cells.Implanting also includes cells grown on solid matrices or prosthetics aswell as cells encapsulated in semi-permeable membranes or barriers. Theterm “implanting” is also intended to include the grafting of cells fromnon-solid tissues such as the hematopoietic system. Such implantingtherefore includes, for example, the direct injection of cells into theblood stream, tissue or abdominal cavity of an individual or theintroduction of the cells into an individual by surgical manipulation.

[0031] As used herein, the term “coexpressing” is intended to mean theexpression of two or more molecules by the same cell. The coexpressedmolecules can be polypeptides or nucleic acids. When referring tonucleic acids, the expression can be, for example, constitutive orinducible. Such nucleic acid sequences can also be expressedsimultaneously or, alternatively, regulated independently. Variouscombinations of these modes of coexpression can additionally be useddepending on the number and function of amino acid or nucleotidesequences being expressed. Those skilled in the art know, or candetermine, what modes of coexpression can be used to achieve aparticular goal or satisfy a desired need.

[0032] As used herein, the term “insulin” is used to mean a polypeptidecapable of stimulating glucose uptake by cells in response to increasedglucose levels. Insulin can correspond to the amino acid sequence or anyportion thereof from a variety of vertebrate species such as human,porcine, equine, rat or bovine so long as the resulting expressedmolecule retains at least one bioactive function such as the stimulationof glucose uptake, glycogen synthesis, amino acid uptake, or proteinsynthesis (see Table 2 below). The term also includes modified forms ofinsulin having amino acid substitutions that enhance or do not greatlydiminish the bioactivity of the polypeptide to stimulate glucose uptakeby cells. Insulin also can include additions or deletions of amino acidresidues so long as it retains insulin bioactivity. A bioactive insulincan therefore have an activity that is similar to wild type insulin oris higher or lower than wild type insulin so long as the bioactiveinsulin stimulates glucose uptake by cells.

[0033] Generally, human insulin has the molecular weight of about 5.8kDa. The human insulin A- and B-chain sequences are provided asexemplary sequences for the insulin polypeptides of the invention (seeFIG. 4). The A-chain of human insulin corresponds to nucleotides 312-374(SEQ ID NO:3) and amino acids 90-110 (SEQ ID NO:4), and the B-chain ofhuman insulin corresponds to nucleotides 117-206 (SEQ ID NO:5) and aminoacids 25-54 (SEQ ID NO:6) of the sequences shown in FIG. 4. Humaninsulin genomic DNA can be found at GenBank accession No. J00265, andhuman insulin cDNA can be found at GenBank accession No. X70508.Nucleotide and amino acid sequences of insulin polypeptides from speciesother than human are known to those skilled in the art. All of thesesequences as well as substantial equivalents and functional fragmentsthereof that maintain insulin bioactivity are included within the term“insulin” as used herein. In general, insulin consists of an A-chainregion disulfide linked to a B-chain region.

[0034] As used herein, the term “proinsulin” is intended to mean aprecursor form of insulin. Proinsulin polypeptides of the invention canbe the amino acid sequence, or portions thereof, corresponding to avariety of vertebrate species such as human, porcine, equine, rat orbovine so long as it contains an A- or B-chain region of insulin, or afunctional fragment thereof. The term includes modified forms ofproinsulin so long as the precursor polypeptide can be processed, ormodified to be processed, into a bioactive form of insulin or into anA-chain or a B-chain region of insulin which is capable of assemblinginto a bioactive form of insulin. The precursor regions of theproinsulin polypeptides can be essentially any amino acid sequence solong as it does not negatively affect the processing of proinsulin intoa bioactive form of insulin, an A-chain, a B-chain, or a functionalfragment thereof. Therefore, the precursor region sequences can be, forexample, the propeptide of insulin such as the proinsulin sequence shownin FIG. 4 or the C-chain region which are found in vertebrate proinsulinmolecules. Alternatively, such precursor regions can be, for example,any of a variety of amino acid sequences, such as linker sequences, thatare not normally found in vertebrate proinsulin molecules. Thenucleotide and amino acid sequences of human proinsulin are recited asSEQ ID NOS:1 and 2, respectively, and are provided as exemplarysequences for the proinsulin polypeptides of the invention (see FIG. 4).Nucleotide and amino acid sequences of insulin polypeptides from speciesother than human are known to those skilled in the art. All of thesesequences as well as substantial equivalents and functional fragmentsthereof, that maintain their ability to be processed into bioactiveinsulin or into an A-chain or a B-chain region of insulin which iscapable of assembling into a bioactive form of insulin are includedwithin the term as used herein. Proinsulin can consist, for example, ofa C-chain connected to the B- and A-chain sequences.

[0035] As used herein, the term “proinsulin cleavage site” is intendedto mean an amino acid sequence that can be recognized by a protease andcleaved, resulting in two or more amino acid fragments. A proinsulincleavage site therefore includes, for example, a sequence within theproinsulin precursor molecule which, upon cleavage by a protease,converts the molecule into bioactive insulin or functional forms of aninsulin A- or B-chain. The term similarly includes cleavage sites thatare not derived from a wild type insulin sequence. For example, acleavage site can be a sequence that is modified or altered so as toallow specific recognition and cleavage by a protease that would notnaturally cleave a wild type proinsulin. Specific examples of proinsulincleavage sites include tetrabasic amino acid sequences such as aArg-Xaa-Lys/Arg/Xaa-Arg motif (SEQ ID NO:7), or dibasic amino acidsequences such as the Arg-Xaa-Lys/Arg-Arg (SEQ ID NO:8). Therefore,essentially any sequence can be used as a proinsulin cleavage site solong as it can be selectively recognized and cleaved by a protease.

[0036] As used herein, the term “protease” is intended to mean apolypeptide capable of specifically recognizing a proinsulin cleavagesite and hydrolyzing a peptide bond. A protease therefore includes anendopeptidase that is capable of specifically recognizing a proinsulincleavage site. A protease also includes the naturally occurringproinsulin cleaving endopeptidases known as PC3 (also known as PC1) andPC2, which normally reside in pancreatic islet β-cells. The termsimilarly includes other subtilisin related enzymes such as furin andPACE4 and the yeast endoprotease Kex 2. Other proteases with known andselective recognition sites are similarly included within the definitionof the term.

[0037] As used herein, the term “glucose-regulated” is intended to meanthe regulation of expression of a nucleic acid sequence by changes inlevels of glucose. For example, glucose-regulated expression includesthe induction of promoter activity by increased levels of glucose. Suchglucose-regulated promoters include, for example, a TGF-α promoterelement, a fibroblast growth factor promoter element, an insulinpromoter element, a PC2 promoter element or a PC3 promoter element. Theterm “glucose” when used in reference to the regulated expression of agene is intended to include both glucose and glucose metabolites so longas such metabolites can cause increased expression from aglucose-regulated promoter. A glucose metabolite includes thoseintermediate products of glucose metabolism such as glucose-6-phosphate,fructose-6-phosphate, glyceraldehyde-3-phosphate, glycerate-2-phosphateand pyruvate.

[0038] The term “glucose” when used in reference to glucose-regulatedexpression is also intended to include an intermediate or product of abiosynthetic pathway that is activated in response to increased levelsof glucose and, therefore, increased glucose metabolism so long as suchintermediates or products can cause increased expression from a glucoseregulated promoter. Such a pathway includes, for example, the hexosaminebiosynthetic pathway where glucosamine-6-phosphate is one intermediatewhich can activate a glucose regulated promoter. Other hexosaminebiosynthetic pathway intermediates include, for example, glucosamine,N-acetyl glucosamine-6-phosphate, N-acetyl glucosamine-1-phosphate,UDP-N-acetyl glucosamine and other hexosamines. An intermediate orproduct of a metabolic pathway that is activated in response toincreased levels of glucose is similarly included within the meaning ofthe term so long as such a metabolic intermediate or product can causeincreased expression from a glucose regulated promoter.

[0039] As used herein, the term “pharmaceutically acceptable carrier” isintended to mean a solution or media which is appropriate foradministration to an individual. Such solutions or media can act tomaintain the stability of compounds and polypeptides and the viabilityof the cells. Pharmaceutically acceptable carriers are well known in theart and include aqueous solutions such as phosphate-buffered saline ormedia. A pharmaceutically acceptable carrier also includes additionalcompounds that act to enhance or increase the ability of the cells toattach or adhere to their in vivo environment. Such compounds caninclude, for example, extracellular matrix molecules such asfibronectin, collagen, laminin, proteoglycans, and fragments thereofcontaining cell adhesion binding sites.

[0040] As used herein, the term “selectable marker” is intended to meana genotypic characteristic of a cell which can be used for identifyingand isolating the cell. A selectable marker includes both endogenousgenotypes as well as genotypes which are produced through the specificmodification of a cell so long as they allow for the identification andisolation of the cell. Selectable markers are well known in the art andinclude, for example, expression of genes which allow for selectionbased on drug resistance, metabolite usage or affinity isolationmethods. For example, an antibiotic resistance gene such as the neomycinphosphotransferase (neo) gene can be used as a selectable marker. Cellsmodified to contain and express the neo gene will be rendered resistantto antibiotic treatment, allowing for selection of these cells. Thebacterial L-histidinol dehydrogenase (hisD) gene which confersresistance to the amino alcohol L-histidinol is also an additionalexample of a selectable marker. The hygromycin resistance gene is yetanother specific example of a selectable marker. The selectable markerneed not confer resistance to a particular treatment but can also be anexpressed molecule that allows for selection of a cell by positiveselection, such as with immunoaffinity beads.

[0041] As used herein, the term “prosthetic graft” is intended to meancells seeded onto a biological material or contained within asemipermeable barrier that is suitable for implantation into anindividual. Such a biological material is any substance which wouldallow for attachment of the cells and allow the cells to remain viableand functional within an individual. A prosthetic graft can consist ofcells seeded onto a ring of polytetrafluoroethylene (PTFE) and thenplaced into an individual. Depending on the level of expression, cellpopulations greater than about 10⁵, preferably greater than about 10⁶,and more preferably greater than about 10⁷ or more cells grown on graftscan provide therapeutic benefit in vivo. A prosthetic graft can alsoconsist of cells contained within a porous membrane or filter and thenplaced into an individual. A prosthetic graft can also include, forexample, cells contained within a biological or synthetic matrix whichcan be implanted into an individual.

[0042] As used herein, the term “hexosamine synthetic pathway enzyme” isintended to mean a molecule which catalyzes the conversion of glucose ora glucose metabolite such as fructose-6-phosphate, to any one of theintermediates or products within the hexosamine biosynthetic pathway.For example, a converting enzyme of this pathway includesglutamine:fructose-6-phosphate amidotransferase (GFA), which catalyzesthe conversion of fructose-6-phosphate to glucosamine-6-phosphate.

[0043] As used herein, the term “three-gene vector” is intended to meana vector comprising elements sufficient to effect the expression ofthree nucleic acid sequences linked on a single vector. The nucleic acidsequences encoding the three genes can be transcribed as separatetranscription units under the control of three separate promoters. Thenucleic acid sequences encoding the three genes can also be transcribedas a single polycistronic transcription unit containing internalribosome entry sites or as a polycistronic transcription unit containingtwo genes and a separate transcription unit encoding the third gene.

[0044] As used herein, the term “isolated” is intended to mean apopulation of cells which are substantially free of contaminants ormaterial as they are normally found in nature. An isolated population ofcells also includes cells that are a subpopulation of a larger group ortype of cells. A “population” as used herein is intended to mean a groupof two or more cells. The cells which make up the population can be ofthe same or different lineage and can be a homogenous or heterogenousgroup of cells.

[0045] The invention provides an isolated population of cells. The cellscontain an expressible nucleic acid encoding proinsulin containing aproinsulin cleavage site. The cells also contain a glucose-regulatedexpressible nucleic acid encoding a protease that is capable of cleavingthe proinsulin cleavage site to produce insulin.

[0046] Insulin is normally secreted by the pancreatic beta (β) isletcells. Mature insulin is derived from the processing of a precursor,proinsulin, in β islet cells. This conversion occurs in secretoryvesicles and is the result of propeptide cleavage by endopeptidases.

[0047] The digestion of nutrients into glucose results in elevated bloodglucose levels. Elevated blood glucose then stimulates insulin secretionby the pancreas, and insulin stimulates the uptake of glucose and itsfurther metabolism for storage and fuel for use by tissues of the body.The expression of proinsulin processing enzymes is also regulated inresponse to elevated blood glucose. In diabetes, glucose homeostasis andmetabolism is disrupted either directly at the level of insulinsecretion or indirectly at the level of tissue responsiveness to insulinactivity. Cells which normally secrete insulin can lose their ability tosecrete a functional insulin peptide, or the biological responses toinsulin secretion can be disrupted.

[0048] The invention is directed to coexpression of a glucose-regulatedendoprotease and proinsulin, which results in secretion of bioactiveinsulin in response to elevated glucose levels and maintenance ofglucose homeostasis by coupling insulin secretion with energymetabolism. The present invention provides cells that functionally mimicthe normal process of glucose-regulated insulin secretion. Therefore,the cells of the invention provide a means of recoupling insulinexpression and its activity to changes in levels of glucose. The cellscan be used in therapeutic methods for the treatment of diabetes.Alternatively, the cells can be used in methods for the diagnosis andstudy of diabetes.

[0049] The cells of the invention are generated by introducing into thecells a vector comprising an expressible nucleic acid sequence encodinga proinsulin containing a proinsulin cleavage site and aglucose-regulated expressible nucleic acid encoding a protease capableof cleaving the proinsulin cleavage site. A proinsulin moleculecontaining a proinsulin cleavage site is one that contains a site whichcan be cleaved by a protease specifically recognizing the site andwhich, upon cleavage, results in a bioactive insulin molecule. The cellscoexpress proinsulin and the cognate protease that recognizes theproinsulin cleavage site so that proinsulin is cleaved to form bioactiveinsulin.

[0050] A nucleic acid sequence of the invention that encodes proinsulincontaining a proinsulin cleavage site can encode, for example, wild typeproinsulin. Wild type bioactive insulin consists of an A-chainpolypeptide that is disulfide linked to a B-chain polypeptide. The wildtype proinsulin molecule contains a C-chain region located between theA- and B-chain regions that aids in the folding of the A- and B-chainsfor correct formation of the disulfide linkages. Wild type proinsulincontains two protease cleavage sites, one between the C- and A-chainsand a second between the C- and B-chains. A nucleic acid sequence of theinvention can therefore encode a wild type proinsulin that is cleavedinto a bioactive insulin.

[0051] A bioactive insulin is a molecule that exhibits one or more ofthe well known activities associated with insulin action. Examples ofthe principle actions of insulin are shown in Table 2. A bioactiveinsulin of the invention exhibits one or more of the activities shown inTable 2, in particular, the activity of increasing glucose uptake. TABLE2 Principal Actions of Insulin. Adipose tissue 1. Increased glucoseentry 2. Increased fatty acid synthesis 3. Increased glycerol phosphatesynthesis 4. Increased triglyceride deposition 5. Activation oflipoprotein lipase 6. Inhibition of hormone-sensitive lipase 7.Increased K⁺ uptake Muscle 1. Increased glucose entry 2. Increasedglycogen synthesis 3. Increased amino acid uptake 4. Increased proteinsynthesis in ribosomes 5. Decreased protein catabolism 6. Decreasedrelease of gluconeogenic amino acids 7. Increased ketone uptake 8.Increased K⁺ uptake Liver 1. Decreased ketogenesis 2. Increased proteinsynthesis 3. Increased lipid synthesis 4. Decreased glucose output dueto decreased gluconeogenesis and increased glycogen synthesis General 1.Increased cell growth

[0052] The invention employs nucleic acids encoding proinsulin that canbe cleaved into a bioactive insulin. When using a wild type proinsulin,the expressible nucleic acid of the invention contains the sequencescorresponding to the A- and B-chains of insulin, directly linked in cison a single vector and separated by a sequence encoding for a C-chainregion. In this case, the proinsulin molecule can contain at least twocleavage sites, one between the C- and A-chains and a second between theC- and B-chains. However, additional cleavage sites within the C-chaincould also be constructed, if desired, for example, to increase theefficiency of cleavage. Such a nucleic acid sequence encoding wild typeinsulin consists of a single, contiguous A-C-B chain sequence that istranscribed and translated as a single product and converted tobioactive insulin.

[0053] In addition to the arrangement of A, C and B chains as found inwild type proinsulin, the expressible nucleic acid sequence can alsoencode a proinsulin comprising an A and B chain separated by a linkersequence. As found in wild type insulin, a cleavage site is insertedbetween the A chain and the linker and the B chain and the linker sothat, upon expression of the appropriate protease, the proinsulin iscleaved into A- and B-chains that can associate to form a bioactiveinsulin. The linker sequence can be, for example, a size thatfacilitates correct pairing of the disulfide bonds that connect the A-and B-chains of insulin. The linker sequence can also be as small as asingle cleavage site that separates the A- and B-chains and that, uponcleavage with a protease, releases the A- and B-chains for assembly intoactive insulin.

[0054] The proinsulin of the invention can be from a variety ofvertebrate species and can be a portion of a proinsulin sequence so longas the translated nucleotide sequence can be processed into an insulinmolecule that stimulates glucose uptake by a cell. The insulin moleculeis processed, for example, by cleavage at a proinsulin cleavage sitethat releases an A- and B-chain so that they form an active insulinmolecule. A proinsulin cleavage site can be in any region of theproinsulin molecule so long as cleavage at that site will not abolishthe activity of the resulting insulin molecule.

[0055] An expressible nucleic acid encoding proinsulin containing aproinsulin cleavage site can be constructed using methods well known inthe art. An exemplary expressible nucleic acid sequence encodingproinsulin containing a proinsulin cleavage site is provided herein asSEQ ID NO:2. Methods for constructing an expressible nucleic acidsequence are known in the art, for example, as described by Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989)) and Ausubel et al. (CurrentProtocols in Molecular Biology, John Wiley & Sons, New York (1998)). Forexample, a nucleic acid sequence encoding proinsulin containing aproinsulin cleavage site can be obtained using polymerase chainreaction. A tissue or cell line from the appropriate organism can beused to amplify insulin or proinsulin sequences. Once a proinsulinsequence has been obtained, the native cleavage site can be used.Alternatively, a cleavage site can be inserted at the appropriateposition in the sequence, for example, using site-directed mutagenesisas described below.

[0056] Insulin is secreted by pancreatic β cells to regulate glucoselevels and is directed to the secretory pathway by a signal sequence.The proinsulin of the invention similarly has an appropriate signalsequence for directing proinsulin to the secretory pathway. The signalsequence can be a signal sequence found in wild type insulin or can beany signal sequence that directs proinsulin to the secretory pathway.Appropriate signal sequences sufficient to direct proinsulin to thesecretory pathway are well known in the art and can be derived fromessentially any protein that is processed in the secretory pathway.

[0057] The above-described proinsulin molecules are expressed as asingle transcription and translation unit, which is cleaved to produceinsulin. However, the invention also provides a proinsulin where the A-and B-chains are expressed as separate transcription units. Whenexpressed as separate transcription units, the translation product ofboth the A- and B-chains contains signal sequences sufficient to directthe A- and B-chains to the secretory pathway. For example, thetranslation product of the separately expressed A- and B-chains can eachhave a signal sequence for directing the A- and B-chains to thesecretory pathway so that the A- and B-chains can associate to form abioactive insulin. The separate transcription units for the A- andB-chains can encode a translation product containing a protease cleavagesite that, upon cleavage, releases the A- and B-chains. However, whenexpressed as separate transcription units, the proinsulin translationproducts need not contain a proinsulin cleavage site. Instead, thetranslation products of the transcription units can directly associateinto a bioactive insulin. Expression of A- and B-chains as separatetranscription units can be useful in the invention even though theformation of bioactive insulin is less efficient than that of proinsulincleaved by a protease so long as a sufficient level of bioactive insulinis formed to mediate the desired effect of regulating the level ofglucose.

[0058] The separate A- and B-chain transcription units can be encoded onthe same expression vector or can be encoded on separate expressionvectors. Separate expression vectors can be used where each vectorcontains a portion of the proinsulin encoding sequence so long ascoexpression of the A- and B-chains encoded by the vectors results inassembly of the A- and B-chains into a bioactive insulin. For example,two expression vectors can be used, each containing a nucleic acidsequence encoding either the A- or B-chain of insulin.

[0059] Nucleic acids encoding proinsulin containing a proinsulincleavage site require coexpression of a corresponding protease thatrecognizes the proinsulin cleavage site. In normal individuals,processing of bioactive insulin from wild type proinsulin occurs in thesecretory pathway of β cells. Therefore, wild type proinsulin orproinsulin containing a wild type proinsulin cleavage site expressed ina β cell can be cleaved by the native endopeptidases PC1, PC2 and PC3.

[0060] However, other cell types are also capable of processingmolecules through the secretory pathway. For example, a nucleic acidencoding proinsulin containing a proinsulin cleavage site can beintroduced into a non-β cell of the desired cell type that has secretorypathway processing machinery. In this case, the proinsulin cleavage siteis engineered to be recognized by an endogenous protease in the cell,allowing cleavage of proinsulin by a protease expressed endogenously inthe cell.

[0061] Alternatively, instead of using endogenous proteases,coexpression of a protease with a nucleic acid encoding proinsulincontaining a proinsulin cleavage site can be achieved by introducing anexogenous protease into the cell. For example, an exogenous protease canbe introduced into a cell using an expression vector that isco-transfected or separately transfected with an expression vectorcontaining a nucleic acid encoding proinsulin containing a proinsulincleavage site. Expression of an exogenous protease can be advantageousin that, instead of relying on the activity of an endogenous protease, avariety of sequence specific proteases can be coexpressed with aproinsulin containing the corresponding cleavage site. Various proteasesand methods for introducing corresponding cleavage sites are describedbelow.

[0062] The coexpression of proinsulin and the protease can be achievedusing a variety of expression vectors. The proinsulin and the proteasecan be expressed on the same expression vector or on separate expressionvectors so long as coexpression of proinsulin and the protease encodingsequences results in expression of a protease that is capable ofcleaving the proinsulin into a bioactive insulin molecule in response toincreased levels of blood glucose.

[0063] The coexpression of proinsulin and protease need not beco-regulated at the transcriptional level so long as the protease andproinsulin polypeptides are coexpressed so that the protease can cleaveproinsulin into peptides that can associate to form a bioactive insulin.Coexpression can be the result of inducible expression of one or bothencoding sequences or constitutive expression of one or both sequencesor combinations thereof. For example, proinsulin can be constitutivelyexpressed while the protease is expressed under glucose regulation. Thefibronectin promoter element is one example of a constitutive promoterthat can maintain expression of proinsulin over a sustained period oftime. By expressing a molecule constitutively, the time for respondingto increased glucose levels is dependent on the synthesis of only onemolecule rather than two, resulting in a more rapid response.Furthermore, by having either protease or proinsulin expressionresponsive to glucose, the amount of bioactive insulin produced isregulated by the level of glucose.

[0064] The methods of the invention generally employ vectors containinga nucleic acid encoding a glucose-regulated protease. However, a vectorof the invention can comprise a glucose-regulated proinsulin and aconstitutively expressed protease. In addition, if proinsulin A- andB-chains are expressed as separate transcription units and thetranslation products lack a protease cleavage site, the proinsulin genecan be controlled by a glucose-regulated promoter. Regardless of whichexpression element is glucose regulated or constitutively expressed, theresultant translation product is capable of forming bioactive insulinthat is regulated by glucose levels.

[0065] Glucose-regulated expression of a molecule of interest such as aprotease can be achieved using a variety of promoter elements thatcontrol expression of a downstream gene in response to changes in levelsof glucose. Elements that are responsive to glucose are inducible inthat they generally exhibit low activity in the absence of glucose andare up-regulated in the presence of increased glucose. For example, itis known that TGF-α promoter activity is responsive to glucose (McClainet al., Proc. Natl. Acad. Sci. USA 89:8150-8154 (1992); and Raja et al.,Mol. Endocrinol. 5:514-420 (1991)). Also, sequences from otherglucose-responsive promoter elements can be used such as promoterelements from wild-type insulin, fibroblast growth factor (FGF),epidermal growth factor (EGF), PC2 or PC3 genes (Sander et al., Proc.Natl. Acad. Sci. USA 95:11572-11577 (1998)). Other glucose responsivepromoter elements can be found, for example, in genes for acetyl-CoAcarboxylase, 6-phosphofructo-2-kinase, and L-type pyruvate kinase (Zhangand Kim, Arch. Biochem. Biophys. 15:227-232 (1997); Dupriez andRousseau, DNA Cell Biol. 16:1075-1085 (1997); Antoine et al., J. Biol.Chem. 272:17937-17943 (1997); and Kennedy et al., J. Biol. Chem.272:20636-20640 (1997)).

[0066] To generate a glucose-regulated expressible nucleic acid encodinga protease, the entire promoter sequence can be inserted immediately 5′of the protease sequence in an expression vector. Alternatively, asequence corresponding to a portion of the glucose-responsive promotersequence can be used so long as the chosen region contains at least theminimal sequences sufficient for glucose-regulated stimulation oftranscriptional activity. A nucleic acid encoding a protease underglucose-regulation can be constructed using methods well known in theart as described, for example, by Daniels et al. (Mol. Endocrinol.7:1041-1048, (1993)).

[0067] Additional glucose-regulated promoter or enhancer elementssuitable for use in the invention can be identified and tested usingmethods well known in the art and current information availableregarding activity of known promoter elements. Specific sequencesresponsible for glucose induced activation of gene transcription by theinsulin promoter have been mapped and methods for the identification ofsuch sequences are described by Odagiri et al. J. Biol. Chem.,271:1909-1915 (1996). Additionally, elements from other promotersequences that ate inducible by glucose can be used.

[0068] To identify additional glucose-regulated promoter elements, asequence corresponding to the glucose-responsive region of a promotercan be linked 5′ to a reporter sequence. Luciferase or β-galactosidaseare exemplary reporter gene sequences that can be used.Glucose-responsive induction of the promoter element can be confirmed invitro using cells that have been modified to express the testpromoter/reporter gene sequence. Promoter elements that areglucose-responsive will result in a higher level of detectable reportergene product, for example, luciferase or β-galactosidase, in cellscultured in the presence of high glucose with low or no expression inlow glucose. Promoter elements with altered activity such as enhancedactivity in response to changes in glucose can also be identified byusing such a reporter system. New glucose-responsive promoter elementscan be identified by testing the ability of the element to directincreased expression of a reporter gene in response to increased or highlevels of glucose. Elements identified as glucose-responsive can then beused to regulate insulin expression in a glucose-regulated manner.

[0069] It is understood that a glucose-regulated promoter is one that isresponsive to increased glucose levels as a positive regulator ofoperationally linked downstream genes. The glucose-regulated promoter ofthe invention is responsive to increased glucose levels or to increasedlevels of metabolites that result from increased glucose levels. Aglucose-regulated promoter useful in the invention can therefore beresponsive to a metabolite of glucose that is positively correlated withthe level of glucose. Regardless of the mechanism of regulation of aglucose responsive promoter, a promoter is considered to be aglucose-regulated promoter if it increases expression of downstreamgenes in response to increased glucose levels.

[0070] In addition to the use of a glucose-regulated promoter to controlexpression of an operationally linked gene, the invention is alsodirected to the use of additional mechanisms to enhance the productionof glucose-responsive bioactive insulin. Increases in glucose levelslead to a variety of physiological responses that result in productionof numerous metabolic products. Since the invention is directed toincreasing insulin in a glucose-dependent manner, any metabolic productthat is correlated with increased levels of glucose can be used as asensor for increases in glucose levels. In addition, these metabolicproducts can also be used to enhance the sensitivity to glucose whencombined with a glucose-regulated promoter.

[0071] For example, enzymes in the hexosamine synthetic pathway catalyzethe conversion of glucose or a glucose metabolite such asfructose-6-phosphate, to any one of the intermediates or products withinthe hexosamine biosynthetic pathway. One converting enzyme of thispathway is glutamine:fructose-6-P amidotransferase (GFA), whichcatalyzes the conversion of fructose-6-phosphate toglucosamine-6-phosphate. Because the hexosamine synthetic pathway isstimulated by glucose due to increased availability of substrate, theactivity of enzymes in this pathway such as GFA can be used to increasesensitivity to glucose. For example, glucosamine, the product of thehexosamine synthetic pathway enzyme GFA, was shown to be a strongerinducer of TGFα promoter activity than glucose (Daniels et al., Mol.Endocrinol. 7:1041-1048 (1993); McClain et al., Proc. Natl. Acad. Sci.USA 89:8150-8154 (1992); Raja et al., Mol. Endocrinol. 5:514-520(1991)). Therefore, expression of a hexosamine synthetic pathway enzymesuch as GFA can be used to increase the activity of a glucose-regulatedpromoter such as the TGFα promoter. However, it is understood that anyactivity associated with glucose levels, including enzymatic activity ofa glucose metabolic pathway, can be used in methods and cells of theinvention to sense glucose or enhance the response to glucose.

[0072] The invention additionally provides a population of cellscomprising an expressible nucleic acid encoding proinsulin containing aproinsulin cleavage site, a glucose-regulated expressible nucleic acidencoding a protease and a hexosamine biosynthetic pathway enzyme. Forexample, the invention provides an isolated population of cellsexpressing proinsulin, a glucose-regulated protease, andglutamine:fructose-6-phosphate amidotransferase (GFA). Such cells haveenhanced sensitivity to glucose levels due to increased glucosaminelevels in response to increased glucose levels. Increased glucosaminelevels further activate the glucose-regulated promoter controllingprotease expression. Such cells can be advantageously used to provideglucose-dependent insulin expression in a diabetic patient.

[0073] The methods of the invention are directed to treating orpreventing diabetes in an individual by providing to the individual asupply of insulin that is responsive to the level of glucose. In oneembodiment, glucose regulated expression of bioactive insulin isachieved using a glucose-regulated promoter. However, the methods of theinvention can also utilize an expression vector that is inducible by amolecule that is coordinated with glucose levels. In an individual,glucose levels increase upon ingestion of food. Therefore,administration of a molecule at about the same time as ingestion of foodresults in correlation of levels of the molecule with increases inglucose levels.

[0074] For example, a promoter element can be an element that isinducible by mechanisms other than glucose or by a regulating moleculethat is not glucose or a glucose metabolite. Glucose-regulated inductionof such an element can be achieved by administering to an individual theregulating molecule at a specific time before, during or followingingestion of a meal. For example, a promoter element controlling aprotease encoding nucleic acid sequence can be activated by theadministration or ingestion of a molecule which specifically activatesthat promoter element. The molecule can be formulated into a drug formfor easy absorption by the intestinal mucosa. The amount and timing ofadministration of the molecule can be readily determined by one skilledin the art by measuring the absorption of the molecule relative toincreases in glucose upon ingestion of food. Glucose-regulatedexpression of insulin is then achieved by administration of the drugwith a meal or at a time before or after the meal that allows levels ofthe molecule to be correlated with glucose levels.

[0075] As described above, methods of the invention employing proinsulincontaining a proinsulin cleavage site require coexpression of aprotease. A variety of proteases can be selected for use in theinvention so long as the protease is able to recognize and specificallycleave a proinsulin cleavage site in the expression vector and generatebioactive insulin. Many bioactive forms of proteins are produced fromprecursor molecules by endoproteolysis. For example, active forms ofmolecules such as melanocyte stimulating hormone, insulin-like growthfactors I and II, adrenocorticotropic hormone, β-endorphin, enkephalinand glucagon are all produced by cleavage with a protease at a specificcleavage site. The specific proteases which convert these describedmolecules can also be used as a protease of the invention. Otherproteases can also be selected for use in the invention if it is capableof recognizing and cleaving at a proinsulin cleavage site.

[0076] The proteases of the invention will generally be endopeptidaseshaving specificity for a peptide sequence rather than recognition of asingle amino acid as a cleavage site. As described above, examples ofproteases useful in the invention include PC1, PC2, PC3, furin, PACE4and Kex2. Other proteases useful in the invention include enterokinase,which recognizes the sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO:9) andcleaves after Lys, or factor Xa, which recognizes the sequenceIle-Glu-Gly-Arg (SEQ ID NO:10) and cleaves after R. When using proteasesthat do not recognize the native proinsulin cleavage site are used, theproinsulin cleavage site is modified to the cognate recognition site forthe protease so that the site can be cleaved by the protease.Essentially any protease with specificity that allows cleavage of aproinsulin cleavage site while retaining biological function of thecleaved proinsulin products can be used in the invention. As describedabove, methods for isolating protease genes are well known in the art(Sambrook et al., supra (1989) and Ausubel et al. supra (1998).

[0077] The protease is coexpressed with proinsulin, and, becauseproinsulin is targeted to the secretory pathway, the protease issimilarly targeted to the secretory pathway. Therefore, the proteasegene product encoded by the vectors of the invention has a signalsequence for targeting the protease to the secretory pathway so thatproinsulin can be cleaved by the protease. As with proinsulin, anysignal sequence that directs the protease to the secretory pathway canbe used.

[0078] Just as a variety of proteases can be selected for use in theinvention, a variety of proinsulin cleavage sites can be selected forinsertion into a proinsulin molecule. A proinsulin cleavage site can beany sequence that is recognized and specifically cleaved by a protease.Selection of a proinsulin cleavage site sequence will depend on theprotease that is used and can be determined by one skilled in the art.

[0079] Proteases can differ in their efficiency of cleavage at aparticular sequence. Therefore, selection of a cleavage site will alsodepend on the level of specificity and cleavage desired. For example,cleavage of the prohormone pro-nerve growth factor to the mature form iscatalyzed by furin at dibasic sites such as Arg-Arg in the regulatedpathway of endocrine cells. However, the endoprotease furin can alsocleave with high efficiency at tetrabasic amino acid sequences recitedas SEQ ID NOS:7 and 8. Furin also cleaves at other tetrabasic amino acidsequences but with lower efficiency. Therefore, any one of the abovedibasic or tetrabasic sites can be inserted into a proinsulin moleculeand used as a protease cleavage site for a protease such as furin.

[0080] The location of a cleavage site in proinsulin depends on theparticular arrangement of proinsulin elements in the expressible nucleicacid encoding proinsulin. For example, the cleavage site can be outsideor within the A- and B-chain regions so long as the cleaved proinsulincan associate to form bioactive insulin that can stimulate glucoseuptake. In a nucleic acid sequence encoding proinsulin elements such asthe A- and B-chains, various arrangements of elements can be used forexpressing a proinsulin containing a cleavage site. For example, thenumber of cleavage sites contained in the proinsulin molecule can varydepending on the particular combination of encoding nucleic acidsequences used for expressing proinsulin, but will generally be at leastone cleavage site, but can be two, three or more cleavages sites.

[0081] A single cleavage site can be used, for example, if an A- andB-chain are separated by a linker containing a single cleavage site.Alternatively, a single cleavage site can be used, for example, if thenucleic acid sequence encodes insulin with an inhibitory sequence thatis cleaved to release bioactive insulin. A single cleavage site can beuseful in the invention even though the formation of bioactive insulinis less efficient than that of proinsulin with two cleavage sites solong as a sufficient level of bioactive insulin is formed to mediate thedesired effect of regulating the level of glucose.

[0082] A proinsulin containing two cleavage sites can be used, forexample, when proinsulin elements are arranged as in wild typeproinsulin such as A-C-B or as in a proinsulin of the structureA-linker-B as described above. The choice of a particular combination ofencoding nucleic acid sequences depends on the particular insulinencoding sequence chosen, the protease chosen, the efficiency with whichthe protease cleaves the proinsulin cleavage site, the efficiency of thenucleic acid sequence for encoding a correctly transcribed andtranslated product and the efficiency of folding of the cleaved moleculeinto a bioactive insulin. Furthermore, the combination of nucleic acidsequences and the number of cleavage sites used can depend on the celltype chosen, particular need or use of the modified cells or ease ofconstruction of the encoding sequence. These determinations can readilybe made by one skilled in the art.

[0083] As described above, proteases and cleavage sites specificallyrecognized by the proteases are well known in the art and a skilledartisan would know the appropriate methods to use to modify a nucleicacid to contain such a sequence and test for efficacy of the proteasefor the cleavage site. Modification of a nucleic acid sequence tocontain a proinsulin cleavage site can be performed by standardmolecular biology techniques known in the art as described, for example,by Sambrook et al., supra (1989) and Ausubel et al. supra (1998). Forexample, a specific proinsulin cleavage site can be constructed usingpolymerase chain reaction and cleavage site sequence-specific primers togenerate a proinsulin cleavage site. Verification that the modified siteis cleaved by the protease can then be performed in vitro using theprotease, appropriate buffers and a sufficient quantity of isolatedtranslation product of the proinsulin sequence. Detection of peptidefragments of appropriate size based on the known location of thecleavage site within the proinsulin sequence can be used to confirm theefficacy of the protease for the cleavage site.

[0084] For example, a proinsulin cleavage site recognized by furin canbe introduced into proinsulin. Sequence-specific primers that correspondto the tetrabasic furin recognition sequence Arg-Xaa-Lys-Arg (SEQ IDNO:11) or any other specific recognition sequence can be introduced intoproinsulin. The cleavage site can be inserted, for example, at the B-Cand C-A junctions corresponding to wild-type proinsulin nucleic acidsequence as described by Groskreutz et al. (J. Biol. Chem. 269:6241-6245(1994)) to produce a proinsulin molecule that contains a proinsulincleavage site.

[0085] The vectors of the invention comprising an expressible nucleicacid encoding proinsulin containing a proinsulin cleavage site and aglucose-regulated protease can be prepared by a variety of methods wellknown to those skilled in the art. The vectors contain transcriptionalregulatory elements that allow expression in the cell type selected forimplantation. For example, the vectors contain constitutive or induciblepromoters such as a glucose-regulated promoter operationally linked, forexample, to a protease so that a glucose-regulated protease is expressedin the cell to effect proinsulin cleavage. Additionally, the vector canencode for proteins that enhance responsiveness to glucose levels suchas a hexosamine biosynthetic pathway enzyme. Expression vectors of theinvention can also contain tissue specific regulatory elements such asenhancers that allow for targeted expression in specific tissues.

[0086] A vector of the invention can also contain a selectable marker. Avariety of selectable markers are well known to those skilled in the art(Sambrook et al., supra (1989); Ausubel et al. supra (1998)). Forexample, the neo gene that encodes for neomycin resistance can beencoded by a vector of the invention. Other selectable markers includehisD and the hygromycin resistance gene.

[0087] As described above, a single vector can encode the expressionelements or the expression elements can be encoded on separate vectors.If it is desired to express two or more elements, the individualelements can be expressed from the same vector or on separate vectors,or any combination of vectors containing one to all of the desiredexpression elements. For example, a single expression vector can expressfour independent expression elements such as proinsulin, a protease,glutamine-6-phosphate amidotransferase, and a selectable marker.Alternatively, each of these four independent expression elements can beexpressed on four separate vectors. Although expression elements can beencoded by separate vectors, the combination of expression elements intoa single expression vector can be advantageous in that only a singlevector need be introduced into a cell.

[0088] Vectors of the invention can be plasmid or viral based vectors.In the case of a viral vector, the vector can contain, for example, atleast one viral long terminal repeat and a promoter sequence upstream ofand operably linked to a nucleotide sequence encoding the gene productof interest, followed by at least one viral long terminal repeat andpolyadenylation signal downstream of the sequence encoding the geneproduct of interest. In the case of a single vector encoding for morethan one gene of interest, each gene of interest can be expressed asseparate transcription units. Alternatively, multiple genes can betranscribed as a single transcription unit that contains internalribosome entry sites that allow expression of the genes from apolycistronic messenger RNA (Adam et al., J. Virol. 65:4985-4990(1991)). Additional nucleic acid sequences can be inserted into thevector using methods well known in the art.

[0089] Representative retroviral vectors suitable for use in theinvention and methods for their design are described for example, inOsborne and Miller, Proc. Natl. Acad. Sci. USA, 85:6851-6855 (1988);Osborne et al., Proc. Natl. Acad. Sci. USA, 92:8055-8058 (1995); Rameshet al., Nuc. Acids. Res., 24:2697-2700 (1996); and Hock et al., Blood,74:876-881 (1989). Other vectors may also be used and are well known inthe art, such as lentiviral vectors, DNA vectors, adenoviral vectors,pseudotype retroviral vectors, Epstein-Barr viral vectors,adeno-associated virus, vesicular stomatitis virus-g (VSV-g), VL30vectors, and liposome mediated vectors.

[0090] In one embodiment, a vector of the invention comprises a nucleicacid sequence encoding a modified human proinsulin containing a furincleavage site (Bell et al., Nature 282:525-527 (1979); Sures et al.,Science 208:57-59 (1980); Chekhranova et al., Mol. Biol. 26:596-600(1992)), a nucleic acid sequence for the protease furin (Creemers etal., Mol. Biol., 11:127-138 (1992)) under the control of aglucose-responsive promoter element from the TGF-α gene (McClain et al.,supra (1992); and Raja et al., supra (1991)) and a nucleic acid sequencefor a selectable marker such as the neo gene.

[0091] An example of a vector that allows further enhancement ofglucose-regulated insulin expression is a vector encodingglutamine:fructose-6-phosphate amidotransferase cDNA (Sayeski et al.,Gene, 140:289-290 (1994)). Such a vector can be used to enhance insulinexpression by co-transfecting the vector with a vector containing aglucose-regulated promoter, resulting in a stronger induction of a geneoperationally linked to a glucose-regulated promoter element.

[0092] Once an expression vector comprising an expressible nucleic acidencoding proinsulin containing a proinsulin cleavage site and aglucose-regulated protease has been constructed, the vector can beverified for expression of a proinsulin molecule that is cleaved andassembled into insulin using methods well known in the art. For example,the sequence can be introduced into a cell that does not express insulinand the culture media assayed for the presence of mature insulin usingan assay such as ELISA or radioimmunoassay (RIA). Alternatively, theexpressed product can be tested for its ability to induce any one of theprincipal actions of insulin such as those listed in Table 2. Forexample, the expressed product can be tested for its ability tostimulate increased transfer of glucose into cultured adipocytes ormuscle cells. Measurement of the amount of transfer into the cells canbe made by using radiolabelled glucose. Alternatively, the expressedproduct can be tested for insulin bioactivity by assessment of increaseduptake of a radiolabelled amino acid by cultured muscle cells.

[0093] The invention provides a cell or population of cells comprisingan expressible nucleic acid encoding proinsulin containing a proinsulincleavage site and a glucose-regulated expressible nucleic acid encodinga protease capable of cleaving the proinsulin cleavage site to produceinsulin. A cell or an isolated population of cells of the presentinvention includes, for example, a cell or population of cells thatnormally do not produce insulin such as non-β islet cells and that aremodified to express and secrete insulin. The cells are engineered toexpress a protease that is regulated in response to changes in glucoselevels so that the modified cells functionally mimic glucose-regulatedinsulin secretion in β islet cells. Moreover, glucose-regulatedexpression of the protease advantageously results in the amplificationof insulin expression over levels of insulin expression that would beexpected from coexpression of the protease without glucose-regulation.The cells are modified so as to coexpress both a precursor form ofinsulin, or proinsulin, and a protease which effects the cleavage of theprecursor into functional subunits that can associate to form bioactiveinsulin.

[0094] Numerous different types of cells can be used to constructmodified cells which functionally mimic glucose-regulated insulinsecretion. Cell types to be selected for generating the modified cellsof the invention are those which are capable of polypeptide synthesisand secretion. With the exception of highly specialized cell types, thelarge majority of cells meet these criteria. For example, red bloodcells which are terminally differentiated cells, have lost their proteinsynthesis ability and are therefore unlikely candidates for the insulinsecreting cells of the invention. However, with the exclusion of the fewcell types that cannot synthesize and secrete a polypeptide such asthose characterized above, essentially all other cell types can be usedfor constructing the modified cell or cell populations of the invention.The actual cell type to be used will, therefore, depend on the intendeduse of the modified cells by those skilled in the art.

[0095] The cell type chosen for modification is selected according tothe biological characteristics of the cell and according to geneexpression criteria well known in the art. For example, objectivecriteria such as the ease of culture efficiency, the ease of geneticmodification and other routine cellular and molecular manipulations canbe used to evaluate and select the cell type for modification. Thosecell types which can be passaged for several generations withoutsubstantial loss in viability are preferable candidates for modificationto glucose-regulated insulin producers. As will be described furtherbelow, such cell types include, for example, both primary cells as wellas cell lines. Additionally, criteria such as the proliferationcharacteristics can also be evaluated for selection of the cell type tobe modified.

[0096] Cell types are additionally selected according the efficiencywith which they can be modified to express proinsulin and aglucose-regulated protease of the invention. Cell types that can bereadily modified and selected for the expression of the introduced genesby any of a variety of methods known in the art are applicable forconstructing the glucose-regulated insulin producing cells of theinvention. Availability of promoter and regulatory elements can also beincluded as a criteria for selecting a particular cell type formodification. Such characteristics and criteria are routine and wellknow to those skilled in the art.

[0097] Various combinations of the above exemplary characteristics aswell as other characteristics can additionally be used for selecting acell type to modify. For example, if the objective is to achieve aparticular level of insulin expression using a relatively small numberof cells, then a cell type which is efficiently modified and can expresshigh levels of glucose-regulated insulin can be selected to achieve thedesired result. In contrast, if cell number is not a limiting factor,then it can be desirable to select the cell type because of favorablegrowth or proliferation characteristics. Additionally, variousexpression elements can be utilized to augment or modulate the level ofproinsulin and protease expression so as to complement advantageouscharacteristics or overcome any deficiencies of the selected cell typesfor modifications. Such criteria and characteristics are well known orcan be determined by those skilled in the art.

[0098] For therapeutic applications, the above cell populations canadditionally be chosen to be implantable in an individual and remainviable in vivo without being substantially rejected by the host immunesystem. Those skilled in the art know what characteristics should beexhibited by cells to remain viable following implantation. Moreover,methods well known in the art are available to augment the viability ofcells following implantation into a recipient individual.

[0099] One characteristic that can be exhibited by the cell or cellpopulation to be implanted is that they are substantiallyimmunologically compatible with the recipient individual. A cell isimmunologically compatible if it is either histocompatible withrecipient host antigens or if it exhibits sufficient similarity in cellsurface antigens so as not to elicit an effective host anti-graft immuneresponse. Specific examples of immunologically compatible cells includeautologous cells isolated from a diabetic individual and allogeneiccells which have substantially matched major histocompatibility (MHC) ortransplantation antigens with the recipient individual. Immunologicalcompatibility can be determined by antigen typing using methods wellknown in the art. Using such antigen typing methods, those skilled inthe art will know or can determine what level of antigen similarity isnecessary for a cell or cell population to be immunologically compatiblewith a recipient individual. The tolerable differences between a donorcell and a recipient can vary with different tissues and can be readilydetermined by those skilled in the art.

[0100] In addition to selecting cells which exhibit characteristics thatmaintain viability following implantation into a recipient individual,methods well known in the art can be used to reduce the severity of ananti-graft immune response. Such methods can therefore be used tofurther increase the in vivo viability of immunologically compatiblecells or to allow the in vivo viability of less than perfectly matchedcells or of non-immunologically compatible cells. Therefore, fortherapeutic applications, it is not necessary to select a cell type fromthe diabetic individual to achieve viability of the modified cellfollowing implantation. Instead, and as described further below,alternative methods can be employed which can be used in conjunctionwith essentially any donor cell to confer sufficient viability of themodified cells to achieve a particular therapeutic effect.

[0101] For example, in the case of partially matched or non-matchedcells, immunosuppressive agents can be used to render the host immunesystem tolerable to engraftment of the implanted cells. The regimen andtype of immunosuppressive agent to be administered will, depend on thedegree of MHC similarity between the modified donor cell and therecipient. Those skilled in the art know, or can determine, what levelof histocompatibility between donor and recipient antigens is applicablefor use with one or more immunosuppressive agents. Following standardclinical protocols, administration and dosing of such immunosuppressiveagents can be adjusted to improve efficiency of engraftment and theviability of the cells of the invention. Specific examples ofimmunosuppressive agents useful for reducing a host anti-graft immuneresponse include, for example, cyclosporin, corticosteroids, and theimmunosuppressive antibody known in the art as OKT3.

[0102] Another method which can be used to confer sufficient viabilityon partially-matched or non-matched cells is through the masking of thecells or of one or more MHC antigen(s) to protect the cells from hostimmune surveillance. Such methods allow the use of non-autologous cellsin an individual. Methods for masking cells or MHC molecules are wellknown in the art and include, for example, physically protecting orconcealing the cells, as well as disguising them, from host immunesurveillance. Physically protecting the cells can be achieved, forexample, by encapsulating the cells within a semi-permeable barrier thatallows exchange of nutrients and macro molecules. Such a barrierprevents contact of host immune cells such as T-cells with the cellscontained within the semi-permeable barrier but still allowsglucose-regulated induction and secretion of insulin into thecirculation system. Encapsulated cells can therefore be used as animplantable device for providing viable insulin producing cells whichare glucose-regulated. The encapsulated cells can be permanentlyimplanted or periodically replaced depending on the cell type used andthe location where the device is implanted. An example of asemi-permeable barrier includes natural or synthetic membranes with apore size that excludes cell-cell contact. Generally, a pore size ofabout 0.22 mm is sufficient to allow exchange of macromolecules such asinsulin and growth factors without allowing immune cells access toimplanted cells. However, other pore sizes can also be used withoutaffecting viability of the glucose-regulated insulin producing cells.Alternatively, antigens can be disguised by treating them with bindingmolecules such as antibodies that mask surface antigens and preventrecognition by the immune system.

[0103] Immunologically naive cells can also be used for constructingglucose-regulated insulin producing cells. Immunologically naive cellsare devoid of MHC antigens that are recognized by a host anti-graftimmune response. Alternatively, such cells can contain one or moreantigens in a non-recognizable form or can contain modified antigensthat faithfully mirror a broad spectrum of MHC antigens and aretherefore recognized as self-antigens by most MHC molecules. The use ofimmunologically naive cells therefore has the added advantages ofcircumventing the use of the above-described immunosuppressive methodsfor augmenting or conferring immunocompatibility onto partially ornon-matched cells. As with autologous or allogeneic cells, suchimmunosuppressive methods can nevertheless be used in conjunction withimmunologically naive cells to facilitate viability of theglucose-regulated insulin producing cells.

[0104] An immunologically naive cell, or broad spectrum donor cell, canbe obtained from a variety of undifferentiated tissue sources, as wellas from immunologically privileged tissues. Undifferentiated tissuesources include, for example, cells obtained from embryonic and fetaltissues. An additional source of immunologically naive cells includestem cells and lineage-specific progenitor cells. These cells arecapable of further differentiation to give rise to multiple differentcell types. Stem cells can be obtained from embryonic, fetal and adulttissues using methods well known to those skilled in the art. Such cellscan be used directly or modified further to enhance their donor spectrumof activity.

[0105] Immunologically privileged tissue sources include those tissueswhich express, for example, alternative MHC antigens orimmunosuppressive molecules. A specific example of alternative MHCantigens are those expressed by placental cells which prevent maternalanti-fetal immune responses. Additionally, placental cells are alsoknown to express local immuno-suppressive molecules which inhibit theactivity of maternal immune cells.

[0106] An immunologically naive cell or other donor cell can be modifiedto express genes encoding, for example, alternative MHC orimmuno-suppressive molecules which confer immune evasivecharacteristics. Such a broad spectrum donor cell, or similarly, any ofthe donor cells described previously, can be tested for immunologicalcompatibility by determining its immunogenicity in the presence ofrecipient immune cells. Methods for determining immunogenicity andcriteria for compatibility are well known in the art and include, forexample, a mixed lymphocyte reaction, a chromium release assay or anatural killer cell assay. Immunogenicity can be assessed by culturingdonor cells together with lympohocyte effector cells obtained from adiabetic individual and measuring the survival of the donor celltargets. The extent of survival of the donor cells is indicative of, andcorrelates with, the viability of the cells following implantation.

[0107] The cells of the invention can originate from essentially anytissue or organ. For primary cells, a tissue should be selected that iseasily accessible and contains cells that exhibit desirable growth andexpression characteristics such as those described above. Additionalconsiderations when selecting a tissue source include choice of a tissuethat contains cells that can be isolated, cultured and modified toexpress insulin in a glucose-regulated manner. Examples of sources oftissues include muscle, liver, or skin tissue, as well as venous andhematopoietic tissue. Therefore, cell types within these tissues thatcan be modified to express insulin in a glucose-regulated manner can beisolated. Such cell types include, for example, muscle (smooth, skeletalor cardiac), fibroblast, liver, fat, hematopoietic, epithelial,endothelial, endocrine, exocrine, kidney, bladder, spleen, stem and germcells. Other cell types are similarly known in the art that are capableof being modified to express insulin in a glucose-regulated manner andcan similarly be obtained or isolated from a tissue source as selectedabove. Although human tissue sources are advantageous for therapeuticpurposes, the species of origin of the cells can be devised fromessentially any mammal, so long as the cells exhibit the characteristicsthat allow for expression of insulin in a glucose-regulated manner.

[0108] The invention also provides a method for producing an isolatedpopulation of insulin-secreting cells by transducing cells with a vectorof the invention and isolating the transduced cells. An isolatedpopulation of cells can be obtained or isolated by a variety of methods,and the selected method will depend on the type, location, and desireduse of the cells. Methods for isolating cell populations are well knownin the art as described below. Alternatively, cells which have beenpreviously characterized and isolated can be obtained from a commercialsource, such as a tissue or cell bank (American Type Culture Collection,Rockville, Md.) and used directly for modification. The isolated cellsshould contain a sufficient or effective number of cells of the desiredtype which can be modified to express insulin in a glucose-regulatedmanner. Moreover, the population of cells can comprise one or more celltypes so long as an effective number can be modified to express insulinin a glucose-regulated manner. Therefore, populations of theglucose-regulated insulin producing cells of the invention can becomposed of a single cell type, all of which are modified to expressinsulin in a glucose-controlled manner, or multiple cell types, whereeach cell type is modified to express glucose regulated insulin.Alternatively, the populations of glucose-regulated insulin producingcells of the invention can be composed of two or more cell types, whereat least one cell type is modified to express insulin in aglucose-regulated manner. Such heterogeneous populations can provideadvantages in therapeutic applications where cell viability of implantedcells is augmented by the presence of accessory cells. A specificexample of such heterogenous cell populations would be those derivedfrom fetal tissue sources.

[0109] Any of the cell types described above can be used to produce thecell or populations of the invention. One example of a cell type that isuseful in prosthetic grafts is a fibroblast cell. Fibroblast cells canbe obtained from a variety of tissue sources such as, for example, skin,liver, muscle or arterial tissue. Fibroblast cells are also advantageousin that they are easily obtained and isolated, easily modified, and canproliferate to higher densities within a graft. For example, a 10 cm²piece of prosthetic graft can contain 10⁸ fibroblast cells, or up to 20layers of cells, and can express glucose-regulated insulin attherapeutic levels.

[0110] Cells to be used in a prosthetic graft can require use of anadherent cell type, which would require use of isolation methods thattemporarily digest or release the cells from their surrounding tissue.For example, smooth muscle cells can be obtained from a segment ofvenous or arterial tissue. The smooth muscle cells can be obtainedfollowing enzymatic digestion in trypsin and collagenase and purified bypositive selection using muscle cell-specific antigens such as vonWillebrand factor (Lejnieks et al., Blood, 92:1-7 (1998). Alternatively,cells can be isolated following digestion and further purified bycentrifugation. The centrifugation can be performed in the presence of agradient such as a sucrose gradient, which would allow for furtherseparation of cell populations based on their density. Methods for theisolation of primary cells from a tissue source are well known in theart (see, for example, Freshney, Animal Cell Culture: A PracticalApproach, 2nd ed., IRL Press at Oxford University Press, New York(1992). Maintenance of the cells prior to modification and implantationcan be as a cell suspension, adherent cell culture or as organ culture.Conditions for the maintenance and culture of primary and clonal cellsare well known in the art.

[0111] Once a cell type has been selected as described above, cellsexpressing proinsulin containing a proinsulin cleavage site and aglucose-regulated protease capable of cleaving the proinsulin cleavagesite are generated by introducing a vector expressing theabove-described nucleic acid sequences into an appropriate cell. Methodsfor introducing such vectors into a cell are well known in the art (seefor example, Osborne et al., supra (1995)). One method of introducing avector into a cell is by transfection of plasmid or DNA vectors.Transfection methods are well known in the art and include, for example,calcium phosphate precipitation, electroporation, liposome-mediatedtransfection, and microinjection as described, for example, in Sambrooket al. supra (1989) and Ausubel et al., supra (1998). Alternatively, aretroviral or adenoviral vector can be transduced into a cell. Methodsfor transduction of retroviral and adenoviral-type vectors are also wellknown in the art and are described further in Example I.

[0112] Following transfection or transduction of cells with vectors ofthe invention, the cells are selected using a selectable marker that iseither on the same vector as the gene of interest or is co-transfectedon a separate vector. Methods of selecting cells for expression of aselectable marker encoded by a transfected vector are well known tothose skilled in the art (see, for example, Ausubel et al. supra(1998)). Following selection, an isolated population of cells expressingthe gene products of interest is obtained.

[0113] Verification that the population of cells expresses proinsulinand protease can be determined using methods well known in the art. Forexample, a population of cells modified to express proinsulin andprotease can be verified for the ability to express insulin in aglucose-regulated manner by assaying the amount of insulin secreted intothe culture media in high glucose as compared to low glucose. The levelof insulin secreted by the population of cells can then be measured byradioimmunoassay or by a functional assay for one of the knownbiological functions of insulin such as those listed, for example, inTable 2. An exemplary functional assay could consist of measurement ofthe rate or quantity of radioactive glucose transfer into a test cell ortissue such as adipose or muscle tissue, or measurement of an increasein fatty acid synthesis in a test cell or tissue when treated with mediafrom the above population of cells being verified. Additional methods ofselecting cells containing and expressing proinsulin and insulin includeNorthern analysis and solution hybridization of mRNA obtained from thecells, in situ hybridization, immunohistology, and immunofluorescenceusing antibodies specific for proinsulin, protease or insulin. Furtherselection of a population of cells suitable for use in the invention canbe performed using in vivo models. For example, the population of cellsof the invention useful for treating diabetes can be verified for theirability to induce glucose homeostasis in diabetic rats treated with thecells as compared to diabetic rats not treated with the cells.

[0114] Once a population of cells has been obtained, the cells can beimplanted directly into a patient, processed as prosthetic grafts,frozen for long-term storage, or maintained in culture prior toimplantation into a diabetic individual, depending on the need. It isunderstood that even a single cell expressing proinsulin and a proteaseis useful in the invention. A single cell can be useful, for example, ifthe level of expression of bioactive insulin produced by the cell issufficient to ameliorate or alleviate a sign or symptom of diabetes oris sufficient to prevent onset of diabetes or reduce the severity of thedisease.

[0115] The invention also provides a method of treating or preventingdiabetes by implanting into an individual cells coexpressing proinsulincontaining a proinsulin cleavage site and a glucose-regulated proteasecapable of cleaving proinsulin to produce insulin. A diabetic individualrequiring glucose-regulated insulin secretion can be treated with theabove-described population of cells by a variety of implantationmethods. An individual suitable for treatment with the cells of theinvention is selected using clinical criteria and prognostic indicatorsof diabetes that are well known in the art. Definite clinical diagnosisof at least one of the symptoms of diabetes or pathologies related todiabetes as described previously herein would warrant administration ofthe cells of the invention. A list of exemplary pathological symptoms isincluded in Table 1.

[0116] An individual at risk of developing diabetes as assessed by knownprognostic indicators such as family history, fasting blood glucoselevels, or decreased glucose tolerance also warrant administration ofcells modified to express proinsulin and protease in a glucose-regulatedmanner. One skilled in the art would recognize or know how to diagnosean individual with diabetes or disregulated glucose uptake and,depending upon the degree or severity of the disease, can make theappropriate determination of when to administer the claimed inventionand can also select the most desirable mode of administration. Forexample, whereas a person with long-standing type 1 disease can requireimmediate implantation of the insulin expressing cells, a person withlong-standing type 2 disease could defer treatment until after there isan indication of a lack of effectiveness of other prescribed treatments.

[0117] A population of cells expressing glucose-regulated insulin can beadministered to an individual that has been determined by one skilled inthe art to require treatment for diabetes for amelioration of theirdisease. The cells can be administered for amelioration of one or moresigns or symptoms of diabetes. For example, a diabetic individual can beimplanted with the cells following diagnosis of the disease. Theimplanted cells will express insulin in response to increased bloodglucose levels such as following ingestion of a meal so that glucosehomeostasis is at least partially restored. An individual that has beeneffectively treated for diabetes will exhibit a reduction in severity ofat least one of the symptoms indicative of the disease followingimplantation of the insulin secreting cells. The reduction in severityof a symptom can be determined and would be apparent to one skilled inthe art.

[0118] Individuals with less severe diabetes can also be implanted withthe insulin expressing population of cells of the invention.Determination of a need for treatment in such individuals can be made byone skilled in the art. For example, a diabetic individual that does notrespond or responds poorly to standard treatment methods can be treatedby methods of the invention. A patient with type 2 disease who has triedunsuccessfully to maintain a long-term decrease in weight or to adhereto an exercise regimen, for example, can be treated for their insulinresistance by implantation of a population of cells of the invention.

[0119] The methods of the invention can also be used to improve theefficacy of other therapies for diabetes. The methods of the inventioncan be used in combination with pre-existing or other methods oftreatment to improve the efficacy or ease of use of the other methods.For example, the insulin expressing cells can be implanted in a patientreceiving daily injections of insulin or a patient using an insulinpump. Implantation of the cells can reduce the frequency of insulininjections in such a patient. A diabetic individual not receivinginsulin therapy but receiving behavioral modification therapy, forexample, diet and exercise to decrease weight, can also be implantedwith the insulin expressing cells of the invention. Implantation of theinsulin expressing cells in such individuals, in combination with aweight reduction and exercise regimen, can decrease the likelihood ofdisease relapse or can ameliorate signs or symptoms of the disease. Theinsulin expressing cells of the invention can also be used to treat adiabetic individual having autoimmune responses against endogenousinsulin secreting cells. Such diabetic individuals are often treated byimmunotherapeutic intervention of the autoimmune response. Theseindividuals can be additionally treated with the population of cellsexpressing glucose-regulated insulin to achieve greater therapeuticefficacy than would be achieved with immunotherapy alone.

[0120] The cell populations of the invention, which expressglucose-regulated insulin, can be administered to the individual toproduce an increase in insulin secretion and thereby effect aglucose-uptake response. Engraftment of the cells allows prolongedglucose homeostasis due to the expression of insulin. An individualsuffering from diabetes can be implanted with a population of insulinexpressing cells, for example, smooth muscle cells engineered to expressproinsulin and a glucose-regulated protease, seeded onto a prostheticgraft. Such an individual could have a fasting blood glucose level ofabout 140 mg/dl or greater. Smooth muscle cells can be obtained from thesame patient by vein biopsy and then transduced with the above describedvectors. Expression of glucose-regulated insulin by the prosthetic graftcan ameliorate symptoms of diabetes in the patient for an extendedperiod of time.

[0121] A population of cells suitable for implantation consists of asize or cell number that is within a range that can be obtained,modified to express insulin in a glucose-regulated manner, andintroduced into an individual. The size of the population of cells issufficient to express quantities of glucose-regulated insulin that is oftherapeutic benefit when implanted in vivo. The size of the abovepopulation of cells is preferably about 10⁸ cells, and can be betweenabout 10⁶ to 10⁸ cells, for example, about 10⁶ or about 10⁷ cells, andcan be less than about 10⁶ cells. Choice of cell number will depend onthe source of the cells, the viability of the cells followingimplantation, and the level of insulin expression required. One skilledin the art will know, using methods well known in the art, how todetermine the appropriate number of cells that produce a therapeuticeffect.

[0122] Implantation of cells of the invention expressingglucose-regulated insulin can be by a variety of routes. In addition toimplantation as a prosthetic graft, a population of cells can also beadministered into an individual directly, such as by direct injectionintravenously, intramuscularly, subcutaneously, intraperitoneally, orinto a tissue or organ site. Cells or compositions to be used for directadministration are obtained and prepared by methods well known in theart and suspended in the appropriate carrier, which can be determined byone skilled in the art. For example, the isolated population of cellscan be infused either directly through a catheter connected to a devicecontaining the cells and the catheter inserted into a vein, or can beinjected directly into a tissue. The cells are injected in apharmaceutically acceptable carrier which is defined above and furtherdiscussed below. The cells can also be administered with othercomponents such as matrix components, fragments or other molecules whichfacilitate adhesion of the cells. The cells can be administered insingle or multiple administrations as necessary to achieve sufficientexpression of therapeutic levels of glucose-regulated insulin.

[0123] Alternatively, the cells can be grown on solid matrices orprosthetics, or encapsulated in semi-permeable membranes or barriersprior to insertion into an individual. The individual treated with thecells can then be monitored for efficacy of the treatment by measurementof levels of insulin that is secreted following ingestion of a meal.This could consist of radioimmunoassay measurement of blood levels ofinsulin following a meal. Alternatively, measurement of fasting bloodglucose levels in the individual following implantation of the cells canbe used to determine efficacy of the treatment. A decreased rate ofglucose disposal as determined by a glucose tolerance test can also beused to verify efficacy of the treatment. Additionally, the alleviationof at least one of the symptoms associated with diabetes can also beused to determine efficacy of the treatment. One skilled in the artwould know the appropriate means of evaluating and diagnosing efficacyof the treatment.

[0124] The cells are encapsulated or grown on materials that arebiocompatible in that they generally will be inert and will not induceor minimally induce an immune response. Such biocompatible materialsinclude, for example, polytetrafluoroethylene (PTFE), surgical gradestainless steel and DACRON.

[0125] The invention can also be used for the prevention of diabetes.For example, a population of cells expressing glucose-regulated insulincan be implanted as a prophylactic into individuals at risk ofdeveloping diabetes or suffering from hyperglycemia. The invention canalso be used, for example, in individuals genetically predisposed todeveloping diabetes or in obese individuals at risk for developinginsulin resistance or disregulated hyperglycemia. These individuals canbe implanted with cells expressing glucose-regulated insulin secretionprior to or during the onset of clinically overt hyperglycemia. Thelatter case can be considered as preventing the disease but can also beconsidered as treating the disease because normal glucose homeostasis isobtained before chronic elevated blood glucose levels are indicated.

[0126] In addition to transfecting cells for implantation into anindividual, the vectors of the invention can also be directlyadministered to an individual for genetic modification, for example, forex vivo and in vivo therapy. The characteristics of a vector useful forex vivo and in vivo therapy is generally similar to the characteristicsof vectors useful for targeting cells to generate a population of cellsexpressing glucose-regulated insulin as described above. Viral vectorsare particularly advantageous for ex vivo and in vivo therapy.

[0127] For example, ex vivo therapy can be carried out essentially asdescribed above except that cells are administered directly to theindividual rather than being encapsulated in a matrix. For example,cells can be isolated from an individual as described above andtransduced with a viral vector encoding genes sufficient to expressglucose-regulated insulin. Methods of ex vivo therapy are well known inthe art, for example, as described by Kay et al., Proc. Natl. Acad. Sci.USA 89:89-93 (1992); Chowdhury et al., Science 254:1802-1805 (1991); andGrossman et al., Nature Genetics 6:335-341 (1994).

[0128] The use of a viral vector is particularly advantageous for exvivo and in vivo therapy because viruses typically infect and propagatein specific cell types. Moreover, the natural specificity of viruses forspecific tissues or cell types can be used to target a nucleic acidmolecule encoding proinsulin in vivo to a particular tissue or limitednumber of tissues. Furthermore, both viral and non-viral vectors can bemodified with specific receptors or ligands to alter target specificitythrough receptor mediated events.

[0129] Retroviral vectors are particularly useful in methods of theinvention directed to ex vivo and in vivo therapy. As described inExample I, retroviral vectors were used to express therapeutic levels ofinsulin in implanted cells. Retroviral vectors can similarly be used inex vivo and in vivo therapy. Adenovirus and adeno-associated virus canalso be used as vectors for ex vivo and in vivo therapy and areparticularly advantageous if infection of non-dividing cells is desired.Methods of constructing vectors for ex vivo and in vivo therapeutic useare well known in the art as described, for example, by Kay et al.,Hepatology 21:815-819 (1995); Stratford-Perricaudet et al., J. Clin.Invest., 90:626-630 (1992); and Barr et al., Gene Therapy, 2:151-155(1995).

[0130] The vectors of the invention containing an expressible nucleicacid encoding proinsulin containing a proinsulin cleavage site and aglucose-regulated protease can be introduced directly into anindividual. The vector to be administered to an individual can beformulated as a pharmaceutical composition comprising the proinsulin andprotease expressing nucleic acid sequences and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers are well knownin the art and include aqueous solutions such as water, physiologicallybuffered saline, or other solvents or vehicles such as glycols,glycerol, oils such as olive oil or injectable organic esters.

[0131] A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act for example, to stabilize or increase theabsorption of the expressible nucleic acid sequences. One skilled in theart would know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the proinsulin and protease expressingvector and on the particular characteristic of the expression vector,for example, whether the vector is a viral or plasmid vector.

[0132] The pharmaceutical composition also can be incorporated, ifdesired, into oil-in-water emulsions, microemulsions, micelles, mixedmicelles, liposomes, microspheres or other polymer matrices(Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed., CRC Press,Boca Raton, Fla. (1993); Fraley et al., Trends Biochem Sci., 6:77(1981). Liposomes, for example, which consist of phospholipids or otherlipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer. In addition,liposomes are particularly useful because they can encapsulate theexpressible nucleic acid sequences with high efficiency while notcompromising the biological activity of the agent, preferentially andsubstantially bind to a target cell, and deliver the aqueous contents ofthe vesicle into the target cell with high efficiency (see Mannino etal., Biotechniques 6:682 (1988)).

[0133] Targeting of a liposome for delivery of a vector of the inventionto an individual can be passive or active. Passive targeting, forexample, uses the tendency of liposomes to accumulate in cells of thereticuloendothelial system (RES) and in an organ such as the liver,which contains sinusoidal capillaries. The expression vectors formulatedas liposomes can be infused directly into the portal vein of the liverand will effectively modify liver cells to express insulin due to theconcentration of RES cells in the liver and the sinusoidal nature of thecirculatory system in the liver. Active targeting of liposomescontaining an expression vector can be achieved by coupling a specificligand to the liposome. Such ligands include a monoclonal antibody, asugar, a glycolipid or a protein such as a ligand for a receptorexpressed by the target cells. Either method of targeting can beselected depending on the type of cell or location of tissue to bemodified for insulin expression.

[0134] A vector of the invention encoding proinsulin and a protease isadministered in an amount and regimen that will be effective to theindividual. Generally, the dosage will be about that typical foradministration of nucleic acids and can be determined by one skilled inthe art. An effective amount will depend on the degree of severity ofthe disease in the individual and the level of glucose-regulated insulinexpression desired. If the vector is a virus, the vector particles canbe administered in an amount from 1 plaque forming unit to about 10¹⁴plaque forming units, but can also be from about 10⁶ plaque formingunits to about 10¹³ plaque forming units. The viral vector is purifiedto a concentration ranging from about 0.25% to 25%, preferably about 5%to 20% before formulation. After formulation, a dose of about 1 pg to100 ng viral vector is contained in approximately 0.1 ml to 1.0 ml ofthe pharmaceutical composition.

[0135] Administration of a vector containing an expressible nucleic acidsequence to an individual can be as a single treatment or as multipletreatments depending on the level of insulin expression desired or onthe number of cells to be modified. Methods for the delivery of nucleicacid sequences encoding for a polypeptide are known in the art asdescribed, for example, by Felgner et al., U.S. Pat. No. 5,580,859,issued Dec. 3, 1996.

[0136] The level of modification of cells by a vector containing anexpressible nucleic acid sequence encoding proinsulin and a proteaseuseful in the invention is sufficient to result in secretion of atherapeutic level of glucose-regulated insulin. Multiple administrationscan also be performed to increase the proportion of modified cells, toincrease the number of copies of proinsulin and protease per cell, or tomaintain the effective number of modified cells for a desired duration.Efficacy of the in vivo treatment is achieved if at least one of thesymptoms of diabetes is alleviated or reduced. A reduction in severityof a symptom of diabetes in a treated individual can be determined asdescribed previously by one skilled in the art.

[0137] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Expression of Therapeutic Levels of Insulin

[0138] This example demonstrates that a population of non-islet cellscan be modified to express glucose-regulated insulin at levelssufficient to treat diabetes.

[0139] Studies were performed to demonstrate that non-β islet cells canbe modified to express insulin at therapeutic levels. Vascular smoothmuscle cells were transduced with a retroviral vector constructed toexpress human proinsulin I (SEQ ID NO:1) and shown to be capable ofexpressing mature insulin at high levels. Using standard molecularbiology techniques, a retroviral vector LhISN, in which human proinsulincDNA is expressed from the viral LTR promoter/enhancer and theselectable marker neo gene is driven by the SV40 promoter wasconstructed and obtained at a titer of 3×10⁶ cfu/ml. This vector wasbased on constructs previously described for expression of humanadenosine deaminase (ADA) (Hock et al., supra (1989), purine nucleosidephosphorylase (PNP) (Osborne et al., supra (1988), doggranulocyte-colony stimulating factor (G-CSF) (Osborne et al., ClinicalResearch. 41:194A (1994) and rat erythropoietin (Epo) (Osborne et al.,supra (1995). The resulting amount of proinsulin and insulin secreted bythe transduced cells was then determined using radioimmunoassay forinsulin with anti-insulin antibodies which cross react with proinsulin.

[0140] PA 317 mouse packaging cells and primary rat vascular smoothmuscle cells (SMC) transduced with the above vector secreted high levelsof proinsulin. PA 317 packaging cells secreted 26 munits ofproinsulin/day/10⁶ cells and primary rat SMC secreted 92 munits ofproinsulin/day/10⁶ cells.

[0141] In order to effectively treat diabetes using methods of theinvention, a level of insulin sufficient to regulate glucose levels issecreted by a population of cells that can be feasibly implanted invivo. Diabetic BB rats receive 20-40 units of long-acting insulin perday in a single injection. Thus, the therapeutic levels of insulinrequired is within the range of insulin secreted by 10⁶ to 10⁷transduced cells. This cell number is in the range that can be harvestedand implanted in vivo (Lejnieks et al., supra (1996); Osborne et al.supra (1995)). Therefore, an isolated population of non-islet cells canexpress mature insulin at levels sufficient to treat diabetes whenimplanted into a diabetic individual.

[0142] Expression of insulin in a glucose-regulated manner wasdemonstrated by use of a single, three gene retroviral vector constructdesigned to express proinsulin and the protease, furin, which cleavesthe proinsulin into bioactive insulin.

[0143] A three gene retroviral vector, LrIFNTαFu (FIG. 1) is constructedto express rat or human proinsulin I cDNA (purchased from ATCC,Rockville Md.), neomycin phosphotransferase (neo) and murine furin(Creemers et al., supra (1992)). Expression of the furin cDNA isdesigned to be under the control of promoter elements from rat TGF-α(McClain et al., supra (1992)). The rat proinsulin coding DNA is placedin the vector pLXFN. The selectable neo gene (N) is then placed 3′ to afoot-and-mouth disease internal ribosome entry site to give pLIFN(Ramesh et al., supra (1996)). The rat TGFα promoter is placed 5′ to thefurin sequence and the resulting 2.65 kb fragment is placed 3′ to theneo gene to obtain the pLrIFNTαFu vector.

[0144] Amphotropic retroviral vectors are produced from PE 501/PA 317packaging cells (Miller et al., Mol. Cell. Biol. 6:2895-2902 (1986)) andtitered using NIH 3T3 cells (Osborne et al., supra (1988); Osborne etal., Hum. Gene. Ther. 1:31-41 (1990)). Proviral integrity in packagingand primary smooth muscle cells is then determined by Southern analysisand vector plasmids are sequenced with an Applied Biosystems PE 310automated capillary electrophoresis DNA sequencer. Replication competentviruses are then screened by a sensitive S⁺L⁻ assay (Miller et al.,supra (1986); Miller et al., Somat. Cell. Mol. Genet. 12:175-183(1986)). Verification that these retroviral vector constructs expresstherapeutic levels of insulin in response to changes in glucose levelsis then shown using primary diabetic BB rat and rat vascular smoothmuscle cells (SMC).

[0145] Primary diabetic BB rat cells and SMC are infected and selectedin G418 and conditioned media and assayed for levels of mature insulinsecreted in response to increased glucose. An increase in secretedinsulin in response to increasing glucose concentration in the culturefrom normal (5.5 mM glucose) to high (30 mM glucose) shows that thetransduced cells secrete insulin in a glucose-regulated manner. Thelevels of insulin secreted by an implantable number of cells (10⁶-10⁷)is determined by RIA as described above and will be within the rangesufficient for in vivo treatment of diabetes.

[0146] Further enhancement of glucose-regulated insulin expression wasdemonstrated using a vector encoding for proinsulin containing aspecific protease cleavage site. In this approach, a furin cleavablesite was introduced into the human proinsulin cDNA to produce a mutanthuman proinsulin (I*). The retroviral vector, pLI*FNTαFu, which isdepicted in FIG. 1, contains the mutated human proinsulin (I*) in placeof the wild-type human proinsulin encoding sequence.

[0147] Non-islet cells are capable of synthesizing bioactive insulinwhen transfected with a proinsulin cDNA that has been mutated to containfurin cleavable sites. Co-transfection of furin along with the mutatedproinsulin cDNA further augments the levels of mature insulin that isexpressed by the non-islet cells (Yanagita et al., FEBS Letters311:55-59 (1992)). Use of this proinsulin/furin expression system allowsfor a rapid and enhanced glucose-regulated insulin response to elevatedlevels of glucose and which is described and shown below.

[0148] To obtain mutated proinsulin, furin cleavable tetrabasicprocessing sites were initially introduced into human preproinsulin cDNA(Bell et al., supra (1979); Sures et al., supra (1980); Chekhranova etal., supra (1992)). The sequence encoding this site consists of:Arg-Xaa-Lys/Arg-Arg (SEQ ID NO:8) and allows for maximum (100%)furin-mediated enzymatic conversion of proinsulin to bioactive insulin.The resulting mutant preproinsulin cDNA was then placed under control ofLTR in the pLXSN vector to generate pLhI*SN, and a TGFαFu fragment wasinserted 5′ to SV40-neo to give LhI*TαFuSN, where furin was under thecontrol of the TGFα promoter. PA317 virus packaging cells weretransduced and showed a 4.3-fold increased insulin secretion whencultured in high glucose (629 units/ml insulin) as compared to secretionwhen cultured in low glucose (148 units/ml insulin). These resultsdemonstrate that an implantable population of cells can be engineered toexpress therapeutic levels of glucose-regulated insulin and that therate of insulin secretion can be further enhanced by use of aproinsulin/furin expression system due to the inherent ability of furinto catalyze cleavage of multiple substrate molecules.

[0149] Additional augmentation of glucose-regulated insulin expressionwas demonstrated in studies using an additional vector encoding for aglutamine:fructose-6-phosphate amidotransferase (GFA). Increasedexpression of bioactive insulin by an implantable population of cellscan be further achieved by transduction of a second vector containingGFA. Transfection of cDNA for GFA, the rate limiting enzyme in thehexosamine synthetic pathway that involves glucosamine, leads to a 50 to100 fold glucose-sensitive up-regulation in TGFα promoter activity inrat SMC (Sayeski et al., supra (1994); McKnight et al., J. Biol. Chem.267:25208-25212 (1992)). This enhancement is achieved because the TGFαpromoter is 10-fold more sensitive to glucosamine than to glucose (Rajaet al., supra (1991)). Thus, cells can be engineered to be exquisitelyresponsive to glucose by stable integration of GFA, in addition to theproinsulin-furin-neo construct.

[0150] The second transduction step is accomplished using a vectoremploying histidine dehydrogenase (HisD) as a selectable marker andhistidinol as a selective agent (Stockschlader et al., Hum Gene. Ther.2:33-39 (1991)). The vector LGFASHisD is constructed from LSHisD byinsertion of a murine GFA cDNA (Sayeski et al., supra (1994)).

[0151] Primary vascular smooth muscle cells are robust and able totolerate this double infection/selection procedure. An isolatedpopulation of cells at a number which can be feasiblely implanted into adiabetic host (10⁶-10⁷ cells) is transduced with the three-generetroviral vector expressing proinsulin, neo and furin under the controlof TGFα promoter elements and a vector expressing GFA. The cells arethen tested for their ability to convert proinsulin to mature insulin inresponse to changes in glucose level by RIA of mature insulin secretedinto the media at low levels of glucose and at high levels of glucose.Results from the RIA are used to verify that an implantable populationof cells express glucose-regulated insulin secretion at levels that aresufficient for therapeutic efficacy in vivo.

[0152] The results described above demonstrate that non-islet cells canexpress high levels of mature insulin in response to hyperglycemia, thatthis expression is rapid and under tight-glucose regulation, and thatthe insulin levels expressed from a population of cells whose size is inthe range that can be implanted in vivo are levels of insulin havingtherapeutic benefit.

EXAMPLE II Treatment of Diabetic Rats by Insulin Expression fromProsthetic Grafts Implanted in the Stomach

[0153] This example demonstrates that smooth muscle cells transducedwith the glucose-regulated insulin expressing vectors aretherapeutically effective when implanted into diabetic rats.

[0154] Vascular smooth muscle cells transduced with the above-describedvectors are implanted in diabetic BB rats using a prosthetic graftplaced into the stomach wall. Prosthetic grafts are constructed byseeding transduced cells in the procedure as described below.

[0155] Rat smooth muscle cells are isolated from aorta using enzymaticdigestion and characterized by positive staining for muscle cellspecific actins with the HHF35 antibody, while staining negative for vonWillebrand factor (an endothelial cell specific marker). Fortransplantation, Fisher 344 rats are anesthetized and a 3-cm midlineabdominal incision from the xyphoid to the umbilicus is made. Thestomach is temporarily exteriorized and a superficial 0.5 cm incision ismade in the capsule on the cranial face of the stomach body. A smallpocket (0.6 cm diameter) is created under the capsule of the stomachusing blunt dissection and a small PTFE ring is then inserted into thepocket and sutured in place using 5-0 maxon thread. The PTFE rings areplaced under the serosal plane of the stomach. The suture material isdrawn tightly to constrict the ring to a final diameter of 2-3 mm beforefinishing the knot. The fibrous tunic directly overlying the ring iscryofrozen using a steel probe, and the ring is mechanically elevated.

[0156] The inserted graft as described above consists of a ring (4 mminner diameter, 6 mm outer diameter) of polytetrafluoroethylene (PTFE)to retain and provide a niche for the transduced cells (Osborne et al.,supra (1993); Osborne et al., supra (1995); Dale et al., Blood81:2496-2502 (1993)). Smooth muscle cells transduced with theglucose-regulated insulin and GFA expression vectors are injected at 10⁶cells/50 μl media into the center of the ring through a 24-g intravenous(IV) catheter. Animals receive two rings each containing the transducedcells. The diabetic rats are then monitored for insulin and glucoselevels. Rats containing the stomach implants will show a long-termregulated decrease in blood glucose levels to euglycemia, resulting frominsulin secretion in response to hyperglycemia. The use of prostheticgrafts for long-term sustained expression of vector-encoded genes atlevels which achieve therapeutic efficacy can be achieved in vivo andare described below.

[0157] In a study analogous to the rat model of diabetes describedabove, long-term in vivo efficacy of prosthetic grafts expressing anerythropoietin (Epo) gene was verified in rats using the proceduresdescribed above. Animals which had been treated with implants containingcells transduced with the erythropoietin gene showed elevatedhematocrits and demonstrated that retrovirally transduced smooth musclecells allowed sustained expression of the transduced genes over thelong-term (Osborne et al, supra (1995).

[0158] Nine rats received prosthetic grafts containing cells transducedwith a erythropoietin expressing vector. The prosthetic grafts wereimplanted as described above. Following implantation, the prostheticgraft became vascularized and permitted the proliferation and long-termsurvival of the transduced smooth muscle cells. Implantation into thestomach wall protected the transduced cells from ingrowth ofnon-transduced cells. The prosthetic graft became vascularized, thuspermitting the proliferation and long-term survival of transduced smoothmuscle cells. Histological examination of a PTFE ring from one of therats at 1 year showed that the tissue within and around the PTFE graftwas fully integrated, well vascularized and that the transduced smoothmuscle cells were contained within the PTFE ring (Lejnieks et al.,supra(1998)).

[0159] Hematocrits of animals containing Epo-expressing prostheticgrafts rose over 2 months from a mean of 42.6±1.4% pre-surgery to rangebetween 55% to 70% over the 12 month test period (FIG. 2). Alltreated)animals maintained a mean hematocrit of 60%±4.7%. Hemoglobinlevels in these animals rose from a pre-surgery mean of 15.2±0.4 g/dl toa seven week maximum of 22.6±2.0 g/dl with a mean of 20.6±1.4 g/dl. Thehematocrits and hemoglobin levels of control animals (animals treatedwith human ADA encoding vector) remained unchanged. Thus, prostheticgrafts seeded with transduced vascular smooth muscle cells implanted bya PTFE stomach patch sustained gene expression for greater than a year.These results demonstrate and verify that smooth muscle cells can beimplanted in vivo as prosthetic grafts and that the engrafted transducedcells maintain long-term expression of vectors transduced into thesmooth muscle cells at levels sufficient for therapeutic efficacy.

[0160] Efficacy of using prosthetic grafts containing transduced cellsfor sustained vector encoded gene expression was also demonstrated inadditional animal models. Baboons were implanted with prosthetic PTFEgrafts containing autologous vascular SMC (Geary et al., Hum. Gene Ther.5:1213-1218, 1994). Retroviral vectors encoding β-galactosidase (LNPoZ)or a control gene, human purine nucleoside phosphorylase (LPNSN-2), weretransduced into autologous baboon SMC. The SMC were obtained from veinbiopsies. Transduced cells were placed into a collagen solution andseeded onto the luminal surface of the grafts. One LNPoZ-seeded graftand one LPNSN-2-seeded control graft was implanted bilaterally into theaorto-iliac circulation of each of 4 animals. All grafts remained patentuntil they were removed after 3-5 weeks and examined histologically forvector-expressing cells.

[0161] Histological examination of the prosthetic grafts containingβ-galactosidase-vector seeded cells showed positive blue staining withthe X-gal chromogen in all of the animal. No sections from the controlgrafts contained stainable cells. Smooth muscle cells expressing thereporter gene were localized within the graft wall but not in the newlyforming intima or outer capsule of fibrous tissue. These results furtherdemonstrate that a population of cells implanted in vivo via prostheticgrafts provides sustained vector encoded gene-expression. Use of thistype of graft will be effective in providing therapeutic long-termsystemic glucose-regulated insulin secretion in diabetic patients.

[0162] In an additional related animal model, a different method ofobtaining prosthetic grafts was used to implant smooth muscle cellscontaining G-CSF or β-gal encoding vector into dogs. Cells were seededinto the prosthetic graft without using collagen polymerization. Thegrafts containing seeded cells were cultured for 5-7 days prior tosurgical implantation. This resulted in the formation of a mature cellmatrix within the graft which allowed for an increased number of cellsfor implantation. Autologous endothelial cells were also seeded onto thegraft luminal surface 24 hrs prior to surgery to reduce thrombosis. Thegrafts were implanted as two 6 cm shunts in the left and right femoralarteries of dogs. At intervals, 1 cm sections of graft were removed andstained for β-gal activity. A section of graft removed at 7 days showeda wide, uniform distribution of transduced cells (FIG. 3). A graftsection obtained at 11 days showed layers of transduced cellsconstituting a new intimal surface, with adjacent transduced cells inthe graft wall (FIG. 3). This alternative method of cell seeding allowedfor a large increase in the number of transduced cells within the graftmatrix, an even distribution of cells throughout the graft wall, andformation of a new intimal surface by the transduced cells.

[0163] Using the same alternative method of cell seeding describedabove, expression of G-CSF by prosthetic grafts in a dog model of caninecyclic hematopoiesis also resulted in long-term levels of vector encodedgene expression and at therapeutic levels. The interstices of a PTFEgraft were seeded with SMC transduced to express canine G-CSF withautologous non-transduced endothelial cells seeded onto the luminalsurface. Neutrophil counts were monitored before and after implantationof two 5 cm grafts implanted as described above. In one dog, neutrophilsincreased 43% from a mean count of 5,450 neutrophils/μl before surgeryto 7,790 neutrophils/μl after surgery, giving a net increase of 2,340neutrophils/μl over the 88 day observation period. In a second animalobserved for 35 days, neutrophils increased by 50% after surgery ascompared to neutrophil levels before surgery. Responses of thismagnitude may be more than adequate to improve the clinical status ofpatients with cyclic or chronic neutropenia. These results furtherdemonstrate the efficacy of using prosthetic grafts implanted with cellsgenetically modified to supply sustained glucose-regulated insulin fortreatment of diabetes.

[0164] Throughout this application, various publications have beenreferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

[0165] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theintention. Accordingly, the invention is limited only by the claims.

1 11 1 450 DNA Homo sapiens CDS (45)..(377) 1 gctgcatcag aagaggccatcaagcacatc actgtccttc tgcc atg gcc ctg tgg 56 Met Ala Leu Trp 1 atg cgcctc ctg ccc ctg ctg gcg ctg ctg gcc ctc tgg gga cct gac 104 Met Arg LeuLeu Pro Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro Asp 5 10 15 20 cca gccgca gcc ttt gtg aac caa cac ctg tgc ggc tca cac ctg gtg 152 Pro Ala AlaAla Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val 25 30 35 gaa gct ctctac cta gtg tgc ggg gaa cga ggc ttc ttc tac aca ccc 200 Glu Ala Leu TyrLeu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro 40 45 50 aag acc cgc cgggag gca gag gac ctg cag gtg ggg cag gtg gag ctg 248 Lys Thr Arg Arg GluAla Glu Asp Leu Gln Val Gly Gln Val Glu Leu 55 60 65 ggc ggg ggc cct ggtgca ggc agc ctg cag ccc ttg gcc ctg gag ggg 296 Gly Gly Gly Pro Gly AlaGly Ser Leu Gln Pro Leu Ala Leu Glu Gly 70 75 80 tcc ctg cag aag cgt ggcatt gtg gaa caa tgc tgt acc agc atc tgc 344 Ser Leu Gln Lys Arg Gly IleVal Glu Gln Cys Cys Thr Ser Ile Cys 85 90 95 100 tcc ctc tac cag ctg gagaac tac tgc aac tag acgcagcccg caggcagccc 397 Ser Leu Tyr Gln Leu GluAsn Tyr Cys Asn 105 110 cccacccgcc gcctcctgca ccgagagaga tggaataaagcccttgaacc agc 450 2 110 PRT Homo sapiens 2 Met Ala Leu Trp Met Arg LeuLeu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro AlaAla Ala Phe Val Asn Gln His Leu Cys Gly 20 25 30 Ser His Leu Val Glu AlaLeu Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys ThrArg Arg Glu Ala Glu Asp Leu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly GlyGly Pro Gly Ala Gly Ser Leu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly SerLeu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys SerLeu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110 3 63 DNA Homo sapiensCDS (1)..(63) 3 ggc att gtg gaa caa tgc tgt acc agc atc tgc tcc ctc taccag ctg 48 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr GlnLeu 1 5 10 15 gag aac tac tgc aac 63 Glu Asn Tyr Cys Asn 20 4 21 PRTHomo sapiens 4 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu TyrGln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 5 90 DNA Homo sapiens CDS(1)..(90) 5 ttt gtg aac caa cac ctg tgc ggc tca cac ctg gtg gaa gct ctctac 48 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 15 10 15 cta gtg tgc ggg gaa cga ggc ttc ttc tac aca ccc aag acc 90 LeuVal Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 6 30 PRTHomo sapiens 6 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu AlaLeu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro LysThr 20 25 30 7 4 PRT Artificial Sequence SITE (2) any amino acid 7 ArgXaa Xaa Arg 1 8 4 PRT Artificial Sequence SITE (2) any amino acid 8 ArgXaa Xaa Arg 1 9 5 PRT Artificial Sequence Description of ArtificialSequence Consensus Sequence 9 Asp Asp Asp Asp Lys 1 5 10 4 PRTArtificial Sequence Description of Artificial Sequence ConsensusSequence 10 Ile Glu Gly Arg 1 11 4 PRT Artificial Sequence SITE (2) anyamino acid 11 Arg Xaa Lys Arg 1

What is claimed is:
 1. An isolated population of cells comprising anexpressible nucleic acid encoding proinsulin containing a proinsulincleavage site and a glucose-regulated expressible nucleic acid encodinga protease capable of cleaving said proinsulin cleavage site to produceinsulin.
 2. The isolated population of claim 1, wherein said protease isfurin.
 3. The isolated population of claim 1, wherein saidglucose-regulated expressible nucleic acid further comprises atransforming growth factor-α (TGF-α) regulatory element.
 4. The isolatedpopulation of claim 1, wherein said proinsulin and saidglucose-regulated protease are expressed from a single vector.
 5. Theisolated population of claim 4, wherein said vector is a retroviralvector.
 6. The isolated population of claim 4, wherein said vectorfurther comprises a selectable marker.
 7. The isolated population ofclaim 1, wherein said cells express a hexosamine biosynthetic pathwayenzyme.
 8. The isolated population of claim 7, wherein said hexosaminesynthetic pathway enzyme is glutamine:fructose-6-phosphateamidotransferase.
 9. The isolated population of claim 1, wherein saidcells are smooth muscle cells.
 10. The isolated population of claim 1,wherein said proinsulin cleavage site further comprises the followingtetrabasic sequence comprising the amino acids: Arg-Xaa-Lys/Arg/Xaa-Arg(SEQ ID NO:7), wherein Xaa comprises any amino acid.
 11. A three-genevector comprising an expressible nucleic acid encoding proinsulincontaining a proinsulin cleavage site, a glucose-regulated expressiblenucleic acid encoding a protease capable of cleaving said proinsulincleavage site to produce insulin, and a selectable marker.
 12. Thethree-gene vector of claim 11, wherein said protease is furin.
 13. Thethree-gene vector of claim 11, wherein said glucose-regulatedexpressible nucleic acid further comprises a TGF-α regulatory element.14. The three-gene vector of claim 11, wherein said selectable marker isneomycin phosphotransferase.
 15. The three-gene vector of claim 11,wherein said proinsulin cleavage site further comprises a tetrabasicsequence comprising the amino acids: Arg-Xaa-Lys/Arg/Xaa-Arg (SEQ IDNO:7)
 16. The three-gene vector of claim 11 wherein said vector is aretroviral vector.
 17. A method of treating or preventing diabetescomprising implanting into an individual cells coexpressing proinsulincontaining a proinsulin cleavage site and a glucose-regulated proteasecapable of cleaving said proinsulin cleavage site to produce insulin.18. The method of claim 17, wherein said protease is furin.
 19. Themethod of claim 17, wherein said glucose-regulated protease is encodedby a glucose-regulated expressible nucleic acid further comprising aTGF-α regulatory element.
 20. The method of claim 17, wherein said cellsare implanted in prosthetic grafts.
 21. The method of claim 20, whereinsaid prosthetic graft comprises polytetrafluoroethylene.
 22. The methodof claim 17, wherein said proinsulin and said protease are expressedfrom a single vector.
 23. The method of claim 22, wherein said vector isa retroviral vector.
 24. The method of claim 22, wherein said vectorfurther comprises a selectable marker.
 25. The method of claim 17,wherein said cells are administered in a pharmaceutically acceptablecarrier.
 26. The method of claim 17, wherein said cells are smoothmuscle cells.
 27. The method of claim 17, wherein said proinsulincleavage site further comprises a tetrabasic sequence comprising theamino acids: Arg-Xaa1-Lys/Arg/Xaa2-Arg (SEQ ID NO:7) wherein Xaa1 andXaa2 is any amino acid.
 28. A method of treating or preventing diabetescomprising implanting into an individual cells coexpressing proinsulincontaining a proinsulin cleavage site, a glucose-regulated proteasecapable of cleaving said proinsulin cleavage site to produce insulin,and a hexosamine biosynthetic pathway enzyme.
 29. The method of claim28, wherein said protease is furin.
 30. The method of claim 28, whereinsaid glucose-regulated protease is encoded by a glucose-regulatedexpressible nucleic acid further comprising a TGF-α regulatory element.31. The method of claim 28, wherein said hexosamine biosynthetic pathwayenzyme is glutamine:fructose-6-phosphate amidotransferase.
 32. Themethod of claim 28, wherein said proinsulin and said glucose-regulatedprotease are expressed from a first vector and said hexosamine syntheticpathway enzyme is expressed from a second vector.
 33. The method ofclaim 32, wherein said first and second vectors are retroviral vectors.34. The method of claim 32, wherein said first and second vector furthercomprises a selectable marker.
 35. The method of claim 28, wherein saidcells are implanted in prosthetic grafts.
 36. The method of claim 35,wherein said prosthetic graft comprises polytetrafluoroethylene.
 37. Themethod of claim 28, wherein said cells are administered in apharmaceutically acceptable carrier.
 38. The method of claim 28, whereinsaid cells are smooth muscle cells.
 39. The method of claim 28, whereinsaid proinsulin cleavage site further comprises a tetrabasic sequencecomprising the amino acids: Arg-Xaa1-Lys/Arg/Xaa2-Arg (SEQ ID NO:7),wherein Xaa1 and Xaa2 comprises any amino acid.
 40. A method forproducing an isolated population of insulin-secreting cells, comprisingtransducing cells with the three-gene vector of claim 11 and isolatingsaid transduced cells.